U.S. patent application number 17/282807 was filed with the patent office on 2021-12-16 for systems and methods for allergen detection.
The applicant listed for this patent is DOTS Technology Corp.. Invention is credited to J. Efrain Alcorta, Brian Christopher Burke, Todd Glendon Campbell, David Carpenter, Deirdre Ellen Day, Matthew Bernard Dean, Kevin Doherty, David Jennings Dostal, Adi Gilboa-Geffen, Thomas Christopher Hartner, Joel F. Jensen, Gregory J. Kintz, Paul Koh, William Law, Russell C. Mead, Jr., Patrick Murphy, Eric Anthony Robertson, Tyler S. Smith, Stanley Owen Thompson, Nhat Nam Trinh, Valerie Villareal, Alan Lloyd Weeks.
Application Number | 20210389245 17/282807 |
Document ID | / |
Family ID | 1000005850901 |
Filed Date | 2021-12-16 |
United States Patent
Application |
20210389245 |
Kind Code |
A1 |
Gilboa-Geffen; Adi ; et
al. |
December 16, 2021 |
SYSTEMS AND METHODS FOR ALLERGEN DETECTION
Abstract
The present disclosure is drawn to devices and systems for
target detection in samples, particularly allergen detection in
food samples. The allergen detection system includes a sampler, a
disposable analysis cartridge and a detection device with an
optimized optical system. The allergen detection utilizes nucleic
acid molecules as detection agents and detection probes.
Inventors: |
Gilboa-Geffen; Adi;
(Wayland, MA) ; Weeks; Alan Lloyd; (S. Easton,
MA) ; Villareal; Valerie; (Boston, MA) ;
Murphy; Patrick; (Allston, MA) ; Robertson; Eric
Anthony; (San Antonio, TX) ; Day; Deirdre Ellen;
(Winchester, MA) ; Dean; Matthew Bernard; (Far
Hills, NJ) ; Campbell; Todd Glendon; (Holliston,
MA) ; Burke; Brian Christopher; (Mahwah, NJ) ;
Smith; Tyler S.; (Cambridge, MA) ; Hartner; Thomas
Christopher; (Pepperell, MA) ; Thompson; Stanley
Owen; (New Boston, NH) ; Trinh; Nhat Nam;
(Quincy, MA) ; Carpenter; David; (Jaffrey, NH)
; Kintz; Gregory J.; (Santa Cruz, CA) ; Koh;
Paul; (New York, NY) ; Dostal; David Jennings;
(Hanover, NH) ; Doherty; Kevin; (Palo Alto,
CA) ; Jensen; Joel F.; (Redwood City, CA) ;
Law; William; (Palo Alto, CA) ; Mead, Jr.; Russell
C.; (Chapel Hill, NC) ; Alcorta; J. Efrain;
(Austin, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DOTS Technology Corp. |
Natick |
MA |
US |
|
|
Family ID: |
1000005850901 |
Appl. No.: |
17/282807 |
Filed: |
October 4, 2019 |
PCT Filed: |
October 4, 2019 |
PCT NO: |
PCT/US2019/054599 |
371 Date: |
April 5, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62862174 |
Jun 17, 2019 |
|
|
|
62741756 |
Oct 5, 2018 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01L 2300/1822 20130101;
B01L 2200/10 20130101; B01L 2200/026 20130101; B01L 2400/0644
20130101; B01L 2200/0689 20130101; B01L 2200/025 20130101; B01L
2200/04 20130101; B01L 2300/04 20130101; G01N 21/6456 20130101;
B01L 2200/16 20130101; B01L 2300/1827 20130101; B01L 3/502
20130101; G01N 2021/6439 20130101; B01L 2300/0681 20130101; B01L
2300/0663 20130101; B01L 2400/0622 20130101; B01L 2300/087
20130101; B01L 2300/0609 20130101; B01L 2300/025 20130101; G01N
21/6428 20130101 |
International
Class: |
G01N 21/64 20060101
G01N021/64; B01L 3/00 20060101 B01L003/00 |
Claims
1. An assembly for detecting a molecule of interest in a sample
comprising: a sample processing cartridge configured to accept the
sample for processing to a state permitting the molecule of
interest to engage in an interaction with a detection agent; and a
detector unit configured to accept the sample processing cartridge
in a configuration which permits a detection mechanism housed by
the detector unit to detect the interaction of the molecule of
interest with the detection agent, wherein the interaction triggers
a visual indication on the detector unit that the molecule of
interest is detected, wherein the visual indication is by
processing images capturing the interaction of the molecule of
interest with the detection agent.
2. The assembly of claim 1 wherein the molecule of interest is an
allergen.
3. The assembly of claim 1 wherein the detection agent is an
antibody or variant thereof, a nucleic acid molecule or variant
thereof, or a small molecule.
4. The assembly of claim 3, wherein the detection agent is a
nucleic acid molecule that comprises an aptamer derived nucleic
acid sequence that binds to the molecule of interest, or variant
thereof.
5. (canceled)
6. The assembly of claim 1 wherein the sample processing cartridge
comprises: a homogenizer configured to produce a homogenized
sample, thereby releasing the molecule of interest from a matrix of
the sample into an extraction buffer in the presence of the
detection agent; a plurality of separate chambers including a
homogenization chamber, a filtrate chamber, and a detection
chamber; a first conduit to transfer the homogenized sample and
detection agent through a filter system to provide a filtrate
containing the molecule of interest and the detection agent; and a
second conduit to transfer the filtrate to a detection chamber with
a window, wherein the detection mechanism of the detector unit
captures and processes the images of the interaction of the
molecule of interest with the detection agent in the detection
chamber through the window to identify the interaction.
7. The assembly of claim 6 wherein the homogenizer comprises a
rotor and wherein the rotor is powered by a motor located in the
detector unit, wherein the motor is functionally coupled to the
homogenizer when the sample processing cartridge is accepted by the
detector unit.
8. The assembly of claim 7 wherein the sample processing cartridge
further comprises a chamber holding wash buffer for washing the
detection chamber and a waste chamber for accepting outflow
contents of the detection chamber after wash.
9. The assembly of claim 8 wherein the sample processing cartridge
further comprises a rotary valve system for controlling transfer of
the homogenized sample to the filter system, for transfer of the
filtrate to the detection chamber, for transfer of the wash buffer
to the detection chamber and for transfer of contents of the
detection chamber to the waste chamber.
10. The assembly of claim 9 wherein the rotary valve system is
further configured to provide a closed position to prevent fluid
movement in the sample processing cartridge.
11. The assembly of claim 6 wherein the detection chamber includes
a transparent substrate with a detection probe molecule immobilized
thereon, the detection probe configured to engage in a probe
interaction with the detection agent, wherein the interaction of
the molecule of interest with the detection agent prevents the
detection agent from engaging in the probe interaction with the
detection probe, and wherein the transparent substrate further
comprises at least one optically detectable control probe molecule
immobilized thereon, for normalization of signal output measured by
the detection mechanism.
12. (canceled)
13. The assembly of claim 11 wherein the transparent substrate
further comprises two different optically detectable control probe
molecules immobilized thereon, for normalization of signal output
measured by the detection mechanism.
14. The assembly of claim 13 wherein the detection probe and
control probe are immobilized on the transparent substrate with a
checkerboard pattern.
15. The assembly of claim 14 wherein the detection agent includes
an optically detectable fluorescence moiety which is activated when
the probe interaction is engaged.
16. (canceled)
17. The assembly of claim 15 wherein the detection mechanism housed
by the detector unit is a fluorescence detection system with a LED
for excitation of fluorescence which includes a plurality of
optical elements placed within a stepped bore in the detector unit
in either a straight or a folded arrangement, the fluorescence
detection system configured for detection of a fluorescence
emission signal and background signal when the probe interaction is
engaged and subjected to fluorescence excitation.
18. (canceled)
19. The assembly of claim 17 wherein the detector unit further
comprises a camera based detector for capturing the reaction on the
transparent substrate and analyzing fluorescence emission signal
and background signal to identify the probe interaction and
transmit the identity of the molecule of interest, or a source of
the molecule of interest to the visual indication such that an
operator of the assembly is informed of the presence or absence of
the molecule of interest or a source of the molecule of interest in
the sample.
20. The assembly of claim 19 wherein the transparent substrate
comprises a plurality of different detection probes for detection
of a plurality of different detection agents configured to provide
a plurality of different interactions with different molecules of
interest in the sample.
21. The assembly of claim 20 wherein the transparent substrate
further comprises a fluidic panel in connection with the probes for
transfer of the filtrate containing the molecule of interest and
the detection agent to contact with the detection probe and control
probe.
22. The assembly of claim 21 further comprising a sampler, the
sampler comprising a hollow tube with a cutting edge for cutting a
source to generate and retain the sample within the hollow tube and
a plunger for pushing the sample out of the hollow tube and into a
port in the sample processing cartridge.
23. An analytic cartridge for detecting a molecule of interest in a
sample comprising: (a) a first compartment with a homogenizer for
receiving a sample and processing the sample, the homogenizer
configured to produce a homogenized sample, thereby releasing the
molecule of interest from a matrix of the sample into an extraction
buffer in the presence of the detection agent and permitting the
molecule of the interest in the sample to engage in the interaction
with the detection agent; (b) a conduit to transfer the homogenized
sample and detection agent through a filter system to provide a
filtrate containing the molecule of interest and the detection
agent; (c) a second compartment for contacting the filtrate
containing the molecule of interest and the detection agent with
detection probes; the second compartment comprising a transparent
substrate that comprises fluidic channels and a detection chip area
with a detection probe immobilized thereon, the detection probe
configured to engage in a probe interaction with the detection
agent, wherein the interaction of the molecule of interest with the
detection agent prevents the detection agent from engaging in the
probe interaction with the detection probe; (d) a rotary valve
system configured to regulate the transfer of the homogenized
sample and detection agent through the filter system, of the
filtrate to the second compartment, and of wash buffer to the
second compartment and outflow contents from the second compartment
to a waste chamber; (e) a compartment for holding wash buffer for
washing the detection area; and (f) a waste chamber for accepting
outflow contents of the detection chamber.
24. The analytic cartridge of claim 23 wherein the second
compartment comprises a window through which the detection
mechanism of a detector unit analyzes the detection reaction
through the window to identify the interaction of the molecule of
interest with the detection agent in the second compartment.
25. The analytic cartridge of claim 24 wherein the detection chip
area of the transparent substrate further comprises an optically
detectable control probe molecule immobilized thereon, for
normalization of signal output measured by the detection
mechanism.
26. The analytic cartridge of claim 24 wherein the detection chip
area of the substrate further comprises two different optically
detectable control probe molecules immobilized thereon, for
normalization of signal output measured by the detection
mechanism.
27. The analytic cartridge of claim 24 wherein the detection agent
is a nucleic acid molecule comprising an aptamer derived nucleic
acid sequence that binds to the molecule of interest, and an
optically detectable fluorescence moiety which is activated when
the probe interaction is engaged.
28.-29. (canceled)
30. The analytic cartridge of claim 27 wherein the detection probe
is a nucleic acid molecule comprising a nucleic acid sequence that
is complementary to the nucleic acid sequence of the detection
agent.
31. The analytic cartridge of claim 30 wherein the substrate is a
glass chip, or a plastic chip, or a membrane like chip.
32. The analytic cartridge of claim 31 wherein the filter system is
composed of a bulk filter that includes a cotton volume and a
membrane filter.
33. The analytic cartridge of claim 32 wherein the cartridge
further comprises a plurality of fluid flow paths for transfer of
the homogenized sample to the filter system, for transfer of the
filtrate to the transparent substrate, for transfer of the wash
buffer to the detection compartment and for transfer of contents of
the detection compartment to the waste chamber.
34. The analytic cartridge of claim 23 wherein the rotary valve
system is further configured to provide a closed position to
prevent fluid movement in the cartridge.
35.-49. (canceled)
50. A system for detecting a molecule of interest in a sample,
comprising: a corer for collecting a sample suspected of containing
the molecule of interest; a disposable analytical cartridge
configured for processing the sample, thereby permitting the
molecule of the interest in the sample to engage in the interaction
with a detection agent; and a detection device configured for
operating the detection test and measuring the interaction between
the detection agent and the molecule of the interest, wherein the
disposable analytical cartridge comprises: (i) a sample processing
chamber with a homogenizer configured to homogenize the sample with
an extraction buffer in the presence of the detection agent,
thereby permitting the allergen of the interest in the sample to
engage in the interaction with the detection agent, (ii) a filter
system configured to provide a filtrate containing the allergen of
interest and the detection agent, (iii) a transparent substrate
that comprises a plurality of fluidic channels and a detection area
with a detection probe molecule immobilized thereon; the detection
probe configured to engage in a probe interaction with the
detection agent, wherein the interaction of the molecule of
interest with the detection agent prevents the detection agent from
engaging in the probe interaction with the detection probe, (iv) a
detection chamber with an optical window, (v) a chamber holding
wash buffer for washing the substrate and the detection chamber,
(vi) a waste chamber for accepting and storing outflow contents of
the detection chamber after wash, (vii) a rotary valve system and
conduits configured to transfer the homogenized sample and
detection agent through the filter system, to transfer the filtrate
to the detection chamber, and to transfer the wash buffer to the
detection chamber and outflow contents from the detection chamber
to the waste chamber, and (viii) an air flow system configured to
regulate air pressure and flow rate in the cartridge.
51.-52. (canceled)
53. The system of claim 50 wherein the filter system comprises a
bulk filter composed of a gross filter and a depth filter, and a
membrane filter, and a filter cap connected to the rotary valve
system.
54. (canceled)
55. The system of claim 53 wherein the detection agent is a nucleic
acid molecule comprising aptamer derived nucleic acid sequence that
specifically binds to the molecule of interest.
56. (canceled)
57. The system of claim 55 wherein the analytic cartridge further
comprises MgCl.sub.2 lyophilized beads.
58. The system of claim 50 wherein the detection area of the
transparent substrate further comprises one optically detectable
control probe molecule, or alternatively two optically detectable
control probe molecules, immobilized thereon, for normalization of
signal output measured by the detection mechanism.
59. (canceled)
60. The system of claim 50 wherein the transparent substrate is
selected from a glass chip, silica, agarose beads, acrylic glass, a
microwell and a microchip.
61. The system of claim 53, wherein the filter membrane comprises
at least one membrane selected from the group consisting of nylon
membrane, PE, PET, PES (poly-ethersulfone) membrane, glass fiber
membrane, polymers membrane, mixed cellulose esters (MCE) membrane,
cellulose acetate membrane, PTFE membrane, polycarbonate membrane,
PCTE (polycarbonate) membrane and PVDF (polyvinylidene difluoride)
membrane.
62. The system of claim 58 wherein the detection device comprises
an optical system for detecting fluorescence signals from the
detection probe and control probe, wherein the optical system
comprises an excitation optics composed of one Light Emitted Diode
(LED), a collimation lens, a filter and a focus lens; an emission
optics composed of a focus lens, two emission filters, one or more
collection lenses and an aperture; and a camera
63. (canceled)
64. The system of claim 62 wherein the transparent substrate is
aligned with the optical system of the device via the optical
window of the detection chamber.
65.-83. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims priority to U.S. Provisional
Patent Application No. 62/741,756 filed Oct. 5, 2018; and U.S.
Provisional Patent Application No. 62/862,174 filed Jun. 17, 2019;
the contents of each of which are incorporated herein by reference
in their entirety.
FIELD OF THE DISCLOSURE
[0002] The present disclosure is drawn to portable devices and
systems for target detection in samples, for example, allergen
detection in food samples. The disclosure also provides methods for
detecting the presence and/or absence of a molecule of interest in
a sample (e.g., an allergen).
BACKGROUND OF THE DISCLOSURE
[0003] Allergy (e.g., food allergy) is a common medical condition.
It has been estimated that in the United States, up to 2 percent of
adults and up to 8 percent of children, particularly those under
three years of age, suffer from food allergies (about 15 million
people), and this prevalence is believed to be increasing. A
portable device that enables a person who has food allergy to test
their food and determine accurately and immediately the allergen
content will be of great benefit to provide for an informed
decision on whether to consume or not.
[0004] Researchers have tried to develop suitable devices and
methods to meet this need, such as those devices and systems
disclosed in U.S. Pat. No. 5,824,554 to McKay; US Patent
Application Pub. No.: 2008/0182339 and U.S. Pat. No. 8,617,903 to
Jung et al.; US Patent Application Pub. No.: 2010/0210033 to Scott
et al; U.S. Pat. No. 7,527,765 to Royds; U.S. Pat. No. 9,201,068 to
Suni et al.; and U.S. Pat. No. 9,034,168 to Khattak and Sever.
There is still a need for improved molecule detection technologies.
There is also a need for devices and systems that detect allergens
of interest in less time, with high sensitivity and specificity,
and with less technical expertise than the devices used today.
[0005] The present disclosure provides a portable assembly and a
device for fast and accurate detection of an allergen in a sample
by using aptamer-based signal polynucleotides (SPNs). The SPNs, as
detection agents, specifically bind to the allergen of interest,
forming SPNprotein complexes. The complexes are them detected and
measured by a detection sensor. The sensor to capture the SPNs may
comprise a chip printed with nucleic acid molecules that hybridize
to the SPNs (e.g., DNA chip). The detection system may comprise a
separate sampler, disposable cartridges/vessels for processing the
sample and implementing the detection assay, and a detector unit
including an optical system for operating the detection and
detecting the reaction signal. The detection agents (e.g., SPNs)
and sensors (e.g., DNA chips) may be integrated into the disposable
cartridges of the present disclosure. The cartridges, detection
agents and the detection sensors may also be used in other
detection systems. Other capture agents such as antibodies specific
to allergen proteins may also be used in the present detection
systems. Such devices may be used by consumers in non-clinical
settings, for example in the home, in restaurants, school cafeteria
and food processing facilities.
SUMMARY OF THE DISCLOSURE
[0006] The present disclosure provides systems, devices, disposable
cartridges/vessels, optical systems and methods for use in
detection of a molecule of interest (e.g., allergen) in various
types of samples, particularly food samples. The allergen detection
devices and systems are portable and handheld.
[0007] One aspect of the present disclosure is an assembly for
detecting a molecule of interest of in a sample, for example, an
allergen in a food sample. The assembly comprises an analytical
cartridge configured to accept the sample for processing to a state
permitting the molecule of interest to engage in an interaction
with a detection agent. The assembly includes a detector unit
configured to accept the analytical cartridge in a configuration
which permits a detection mechanism housed by the detector unit to
detect the interaction of the molecule of interest with the
detection agent. The interaction triggers a visual indication on
the detector unit that the molecule of interest is present or
absent in the sample. The detector unit may be removably connected
to the analytical cartridge.
[0008] In some embodiments, the assembly may further comprise a
separate sampler configured to collect a sample for detection of
the molecule of interest in the sample. In some embodiments, the
sampler is a food corer. The corer may be operatively connected to
the analytical cartridge to transfer the collected sample to the
cartridge.
[0009] In some embodiments, the analytical cartridge is disposable,
and configured to detect one particular molecule of interest, for
example, one allergen. In other embodiments, the analytical
cartridge may be configured to detect a plurality of molecules of
interest in a sample, for example, a set of allergens.
[0010] In some embodiments, the analytical cartridge comprises a
homogenizer configured to produce a homogenized sample, thereby
releasing the molecule of interest from a matrix of the sample into
an extraction buffer that optionally includes the detection agent.
The analytical cartridge also comprises a first conduit to transfer
the homogenized sample with or without the detection agent through
a filter system to provide a filtrate containing the molecule of
interest, or the complexes of the molecule of interest and the
detection agent, and a second conduit to transfer the filtrate,
making the filtrate to be contacted with a detection probe, thereby
permitting an interaction of the detection agent with the detection
probe. The first and second conduits comprise a plurality of
fluidic paths connecting different parts of the conduits from
transferring the processed sample, buffers, filtrate, detection
agents, waste and other fluids.
[0011] In some embodiments, the analytical cartridge may further
comprise a rotary valve system providing a mechanism for
controlling the transfer of the sample and other fluidic components
(e.g., buffers, filtrate and waste) within the analytical
cartridge. The rotary valve switching system may be further
configured to provide a closed position to prevent fluid movement
in the analytical cartridge.
[0012] In some embodiments, the homogenizer and the rotary valve
system may be powered by motors located in the detector unit when
the analytical cartridge is accepted by the detector unit.
[0013] In some embodiments, the analytical cartridge comprises a
plurality of chambers. The chambers are separate but connected for
operation. As a non-limiting example, the analytical cartridge may
include a sample processing chamber, a detection chamber, a waste
chamber, and optionally a buffer chamber. In some embodiments, the
analytical cartridge may further comprise a separate filtrate
chamber to hold the filtrate and optionally further concentrate the
filtrate prior to the transfer to the detection chamber. In some
examples, the detection chamber comprises a detection sensor and an
optical window. The detection mechanism of the detector unit
analyzes the detection reaction through the optical window to
identify the interaction of the molecule of interest with the
detection agent in the detection chamber.
[0014] In some embodiments, the analytical cartridge comprises a
detection sensor for measuring the interaction between the molecule
of interest and the detection agent. The detection sensor is
included in the detection chamber. In one non-limiting example, the
detection sensor is a separate substrate which includes a plurality
of fluidic channels and a detector chip area. The substrate is also
referred to as a chipannel, wherein the fluidic channels and the
detector chip area are connected. In some examples, the chipannel
is a plastic substrate.
[0015] In some embodiments, the detector chip area within the
chipannel comprises at least one reaction panel and at least one
control panel. In other embodiments, the detector chip area within
the chipannel may comprise one reaction panel and two control
panels. In other embodiments, the chipannel may comprise a
plurality of reaction panels and a plurality of control panels.
Optionally, the detector chip area further comprises one or more
fiducial spots that guide image processing by an imaging mechanism
(e.g., a camera) of the detector unit. Any suitable fiducial object
may be spotted as a fiducial marker for reference.
[0016] In some embodiments, the detector chip area within the
chipannel comprises a detection probe molecule immobilized on the
reaction panel. The detection probe is configured to engage in a
probe interaction with the detection agent. An interaction of the
molecule of interest with the detection agent prevents the
detection agent from engaging in the probe interaction with the
detection probe. The detector chip area within the chipannel may
further include an optically detectable control probe molecule
immobilized on the control panel(s), for normalization of signal
output measured by the detection mechanism.
[0017] In one preferred embodiment, the chipannel is a plastic chip
wherein the reaction panel is printed with a nucleic acid-based
detection probe that comprises a nucleic acid sequence
complementary to nucleic acid sequence of the detection agent and
wherein the control panel is printed with nucleic acid based
control probe molecule that does not bind to the molecule of
interest or the detection agent.
[0018] The analytical cartridge may further include a chamber
storing wash buffer for washing the detection chamber and a waste
chamber for accepting outflow contents of the detection chamber
after washing. In some embodiments, the series of bridging fluid
conduits may comprise: (a) a fluid connection between the wash
buffer chamber and the detection chamber; and (b) a fluid
connection between the detection chamber and the waste chamber.
[0019] In some embodiments, the filter in the analytical cartridge
is a filter assembly comprising a bulk filter and a membrane
filter. The bulk filter may comprise a gross filter and a depth
filter. In some embodiments, the filter assembly may further
comprise a filter cap that can lock the rotary valve.
[0020] In some embodiments, the molecule of interest in the
homogenized sample may be brought in contact with the detection
agent prior to the molecule of interest and detection agent in
contact with the detector probe. The contact of the molecule of
interest and detection agent may occur in the extraction buffer
during homogenization, or in the filter during the filtration, or
in the filtrate chamber. In some embodiments, a MgCl2 deposit is
prestored in the filter or in the filtrate chamber.
[0021] In some embodiments, the analytical cartridge may comprise a
data chip unit configured for providing the cartridge
information.
[0022] In some embodiments, the assembly of the present disclosure
comprises a detector unit that is operatively connected to an
analytical cartridge. In some embodiments, the detector unit of the
assembly comprises a detection mechanism to measure detection
signals, i.e., the interaction between the detection agent and
detector probe. As a non-limiting example, the detection mechanism
is an imaging system, such as a camera for fluorescence
imaging.
[0023] In some embodiments, the detector unit of the assembly
comprises an external housing that provides support for the
components integrated for operating a detection reaction and
measuring detection signals, of the detector unit and for accepting
the analytical cartridge. In accordance with the present
disclosure, the components for operating a detection reaction and
measuring detection signals include motors for driving and
controlling the homogenization, and controlling the rotary valve;
pump driving and controlling the fluidic flow of the processed
sample, the filtrate, buffers and waste in the compartments of the
analytical cartridge; an optical system for detecting and
visualizing a detection result; and a display window.
[0024] In some embodiments, the optical system may comprise
excitation optics and emission optics and an optical reader. The
optical system is modified for detecting signals from the detector
chip area of the chipannel within the cartridge.
[0025] In other embodiments, the optical system may comprise a
camera sensor (e.g., a CCD camera and a sCMOS camera) to generate
images of a detection reaction of the detector chip area of the
chipannel. The images are then processed to indicate the detection
results.
[0026] In some embodiments, the detection assembly may comprise a
user interface that may be accessed and controlled by a software
application. The software may be run by a software application on a
personal device such as a smartphone, a tablet computer, a personal
computer, a laptop computer, a smartwatch and/or other devices. In
some cases, the software may be run by an internet browser. In some
embodiments, the software may be connected to a remote and
localized server referred to as the cloud.
[0027] In one non-limiting embodiment of the present disclosure, a
detection assembly comprises an analytical cartridge that is
configured to be a disposable test cup or cup-like container, a
detector unit comprising a docket for accepting the test cup, and
an optional sampler. The disposable test cup or cup-like container
may be constructed as an analytical module in which a sample is
processed and a molecule of interest in the test sample (e.g., an
allergen) is detected through the interaction with a detection
agent.
[0028] In some embodiments, the disposable test cup or cup-like
container comprises a top cover configured to accept the sample and
to seal the cup or cup-like container wherein the top cover
includes a port for accepting the sample and at least one breather
filter that allows air in; a body part configured to process the
sample to a state permitting the molecule of interest to engage in
an interaction with the detection agent; and a bottom cover
configured to connect to the cup body part thereby forming a
detection chamber with an optical window at the bottom of the test
cup, and to provide the connecting surface to a detector unit. The
exterior of the bottom cover comprises a plurality of ports for
connecting a plurality of motors located in the detector unit to
operate the homogenizer, the rotary valve system and the flow of
the fluids. The optical window of the detection chamber is
connected to the detection mechanism in the detector unit. In some
embodiments, the test cup or cup-like container further comprises a
detection sensor such as a transparent substrate with detection
probes immobilized thereon. The transparent substrate is a
chipannel comprising a detection chip area with nucleic acid based
probes immobilized thereon and fluidic paths.
[0029] In one non-limiting embodiment of the present disclosure,
the disposable test cup comprises (a) a first compartment with a
homogenizer for receiving a sample and processing the sample; the
homogenizer configured to produce a homogenized sample, thereby
releasing the molecule of interest from a matrix of the sample into
an extraction buffer in the presence of the detection agent and
permitting the molecule of the interest in the sample to engage in
the interaction with the detection agent; (b) a second compartment
for contacting the filtrate containing the molecule of interest and
the detection agent with detection probes; the second compartment
comprising a chipannel that comprises a plurality of fluidic
channels and a detection chip area with the detection probes
immobilized thereon; (c) a conduit to transfer the homogenized
sample and detection agent through a filter system to provide a
filtrate containing the molecule of interest and the detection
agent; (d) a rotary valve system configured to regulate the
transfer of the homogenized sample and detection agent through the
filter system, of the filtrate to the second compartment, and of
wash buffer to the second compartment and outflow contents from the
second compartment to a waste chamber; (e) a compartment for
holding wash buffer for washing the detection area; and (f) a waste
chamber for accepting outflow contents of the detection chamber. In
some examples, the detection probe is configured to engage in a
probe interaction with the detection agent, wherein the interaction
of the molecule of interest with the detection agent prevents the
detection agent from engaging in the probe interaction with the
detection probe. The fluidic paths within the chipannel transfer
the filtrate, making the filtrate to be contacted with the
detection probe immobilized on the chip area, and transfer the
outflow contents to the waste chamber.
[0030] In some embodiments, the cup top cover further comprises a
layer for providing an identification label.
[0031] In some embodiments, the parts of the disposable test cup
are molded together forming an analytic module.
[0032] Another aspect of the present disclosure relates to a method
for detecting the presence and/or absence of a molecule of interest
in a sample comprising the steps of (a) collecting a sample
suspected of containing the molecule of interest, (b) homogenizing
the sample in an extraction buffer in the presence of a detection
agent, thereby releasing the molecule of interest from the sample
to engage in an interaction with the detection agent comprising a
fluorescent moiety, (c) filtrating the homogenized sample
containing the molecule of interest and the detection agent; (d)
contacting the filtrate containing the molecule of interest and the
detection agent with a detection probe molecule that engages in a
probe interaction with the detection agent, wherein the interaction
of the molecule of interest with the detection agent prevents the
detection agent from engaging in the probe interaction with the
detection probe; (e) washing off the contact in step (d) with wash
buffer; (f) measuring signal outputs from the probe interaction of
the detection probe molecule and the detection agent; and (g)
processing the detected signals and visualizing the interaction
between the detection probe and the detection agent.
[0033] The molecule of interest may include, but is not limited to,
a protein and a variant or fragment thereof, a nucleic acid
molecule (e.g., a DNA or RNA molecule) or a variant thereof, a
lipid, a sugar and a small molecule. In some embodiments, the
molecule of interest may be a protein, or variant and fragment
thereof. In one example, the molecule of interest is an allergen
such as a food allergen. The detection agent may be an antibody or
variant thereof, a nucleic acid molecule or variant thereof, or a
small molecule. In some embodiments, the detection agent is a
nucleic acid molecule comprising a nucleic acid sequence that binds
to the molecule of interest. In one example, the nucleic acid-based
detection agent is a signaling polynucleotide (SPN) derived from an
aptamer comprising a core nucleic acid sequence that binds to the
molecule of interest. The SPN may further comprising a detectable
moiety such as a fluorescent moiety. Accordingly, the detection
probe may comprise a complementary nucleic acid sequence that
hybrids to the free sequence of the SPN.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] FIG. 1 is a perspective view of an embodiment of a detection
system in accordance with the present disclosure comprising a
detection device 100 having an external housing 101 and a port or
receptacle 102 configured for holding the disposable cartridge 300,
a separate food corer 200 as an example of the sampler, a
disposable test cup 300 as an example of the analytical cartridge.
Optionally, a lid 103, execution/action button 104 that allows a
user to execute an allergen detection testing and a USB port 105
may be included.
[0035] FIG. 2A is an exploded perspective view of one embodiment of
the food corer 200 as an example of the sampler.
[0036] FIG. 2B is a perspective view of the sampler assembly
200.
[0037] FIG. 3A is a perspective view of an embodiment of a
disposable test cup 300, comprising a cup top 310, a cup body 320
and a cup bottom 330.
[0038] FIG. 3B is a cross-sectional view of the test cup 300,
illustrating features inside the cup 300.
[0039] FIG. 3C is an exploded view of the disposable test cup
300.
[0040] FIG. 3D is a top (left panel) perspective view and a bottom
(right panel) perspective view of the top cover 312.
[0041] FIG. 3E is an exploded view of the cup top lid 311.
[0042] FIG. 3F is a top perspective view (left panel) and a bottom
perspective view (right panel) of the cup body 320.
[0043] FIG. 3G is a bottom perspective view of the bottom of the
upper housing 320a (upper panel) shown in FIG. 3C and a top
perspective view of the inside of the outer housing 320b (lower
panel) shown in FIG. 3C.
[0044] FIG. 3H is a bottom perspective view (left panel) and a top
perspective view (right panel) of the cup bottom cover 337.
[0045] FIG. 3I is a bottom perspective view of the cup bottom
surface after assembling the bottom 330 and the cup body 320.
[0046] FIG. 4A is an exploded view of one embodiment of the filter
assembly 325.
[0047] FIG. 4B is a cross-sectional perspective view of one
embodiment of the filtrate chamber 322 comprising a filter bed
chamber 431 for placement of the filter assembly 325, a collection
gutter 432 and a filtrate collection chamber 433.
[0048] FIG. 5A is a perspective view of an alternative embodiment
of the cup 300.
[0049] FIG. 5B is an exploded view of the disposable test cup 300
of FIG. 5A (the filter 325 not shown).
[0050] FIG. 5C is a cross sectional perspective view of the cup 300
of FIG. 5A.
[0051] FIG. 6A is an exploded view of an alternative embodiment of
the cup 300.
[0052] FIG. 6B is a top perspective view (right panel) and a bottom
perspective view (left panel) of the cup body 320 of FIG. 6A.
[0053] FIG. 6C is a bottom perspective view of the cup bottom 337
and the bottom of the cup body 320 of FIG. 6A.
[0054] FIG. 6D is an alternative embodiment of the filter assembly
325.
[0055] FIG. 6E is a cross-sectional view of the filter cap 621 when
is assembled with the rotary valve 350.
[0056] FIG. 6F is a perspective view of the rotary valve 350 (upper
panel) and a bottom perspective view of the bottom of the rotary
valve 350 (lower panel).
[0057] FIG. 6G is a bottom perspective view (upper panel) and a top
perspective view (lower panel) of the cup bottom cover 337 shown in
FIG. 6A.
[0058] FIG. 7A is an exploded view of an alternative embodiment of
the cup 300; the cup 300 comprises a chipannel 710.
[0059] FIG. 7B is a perspective view of the chipannel 710 shown in
FIG. 7A.
[0060] FIG. 7C is a bottom perspective view of the chipannel
710.
[0061] FIG. 7D is a bottom perspective view of an alternative
embodiment of the chipannel 710.
[0062] FIG. 7E is exploded view of an alternative embodiment of the
cup 300.
[0063] FIG. 7F is an alternative embodiment of the cup body in
which the filter gasket 623 is overmolded to the cup body.
[0064] FIG. 7G is an alternative embodiment of the rotary valve 350
shown in FIG. 7E.
[0065] FIG. 7H is a cross-sectional view of the cup body 320 shown
in FIG. 7E, showing the overmolded seal 713 to combine several
parts into a single part.
[0066] FIG. 7I is an alternative embodiment of the cup bottom cover
337 with compression coil springs 721.
[0067] FIG. 7J is perspective views of the cup bottom cover 337
shown in FIG. 7I, demonstrating the compression coil springs 721 at
the bottom.
[0068] FIG. 7K is a perspective view of the sacrificial weld bead
material 722 in the bottom of the cup body 320 shown in FIG.
7E.
[0069] FIG. 8A is a top perspective view of the cup body 320
showing features relating to homogenization, filtration (F), wash
(W1 and W2) and waste.
[0070] FIG. 8B is a scheme showing the positions of the rotary
valve 350 during the sample preparation and sample washes.
[0071] FIG. 8C is a diagram displaying the fluid flow inside the
cup 300.
[0072] FIG. 9A is a perspective view of the device 100
[0073] FIG. 9B is a top perspective view of the device 100 in the
absence of the lid 103.
[0074] FIG. 10A is a longitudinal cross-sectional view of the
device 100.
[0075] FIG. 10B is a lateral cross-sectional view of the device
100.
[0076] FIG. 11A is a valve motor 1020 and associated components for
controlling the operation of the rotary valve 350.
[0077] FIG. 11B is a top perspective view of the output coupling
1020 associated with the motor.
[0078] FIG. 12A is a top perspective view of one embodiment of the
optical system 1030.
[0079] FIG. 12B is a side view of the optical system 1030 of FIG.
12A.
[0080] FIG. 13A is an illustration of a chip sensor 333 displaying
the test area and control areas.
[0081] FIG. 13B is a top view of the optical system 1030 and chip
333 showing reflections providing fluorescence measurements of the
chip 333.
[0082] FIG. 13C is a perspective view of another embodiment of the
chip senor 333 or the sensing area 333' of the chipannel 710
displaying one reaction panel 1312, one control panel 1313 and two
fiducial panels 1311.
[0083] FIG. 13D shows an exemplary pattern of the probes in the
reaction panel and control panel of the detection area 333' of the
chipannel 710.
[0084] FIG. 14A shows the optical assembly 1030 in a straight
mode.
[0085] FIG. 14B shows the optical assembly 1030 in a folded
mode.
[0086] FIG. 14C is a cross-sectional perspective view of one end of
the device 100 (right side of FIG. 10B) showing emission optics
1420 including lenses 1421, 1423 and filters 1422a and 1422b placed
in the stepped bore 1480 in the device 100.
[0087] FIG. 15A is a perspective view of another embodiment of the
optical system 1030 comprising an excitation optics 1510, an
emission optics 1520 and a camera-based detector 1530.
[0088] FIG. 15B is a cross sectional view of the optical components
of FIG. 15A as the optical system is configured inside the
detection device 100.
[0089] FIG. 16A is a histogram demonstrating the SPN intensity in a
MgCl.sub.2 lyophilized formulation as compared to the buffer
without MgCl.sub.2 and the MgCl.sub.2 solution.
[0090] FIG. 16B shows the percentage of magnesium recovered from
MgCl.sub.2 formulations deposited on the cotton filter supported on
1 .mu.m mesh.
DETAILED DESCRIPTION OF THE DISCLOSURE
[0091] The foregoing has outlined rather broadly the features and
technical advantages of the present disclosure in order that the
detailed description of the disclosure that follows may be better
understood. Additional features and advantages of the disclosure
will be described hereinafter which form the subject of the claims
of the disclosure. It should be appreciated by those skilled in the
art that the conception and specific embodiment disclosed may be
readily utilized as a basis for modifying or designing other
structures for carrying out the same purposes of the present
disclosure. It should also be realized by those skilled in the art
that such equivalent constructions do not depart from the spirit
and scope of the disclosure as set forth in the appended claims.
The novel features which are believed to be characteristic of the
disclosure, both as to its organization and method of operation,
together with further objects and advantages will be better
understood from the following description when considered in
connection with the accompanying figures. It is to be expressly
understood, however, that each of the figures is provided for the
purpose of illustration and description only and is not intended as
a definition of the limits of the present disclosure. Unless
defined otherwise, all technical and scientific terms used herein
have the same meaning as commonly understood by one of ordinary
skill in the art to which this disclosure belongs. In the case of
conflict, the present description will control.
[0092] The use of analytical devices to ensure food safety has not
yet advanced to the point of fulfilling its promise. In particular,
portable devices based on simple, yet accurate, sensitive and rapid
detection schemes have not yet been developed for detection of the
wide variety of known allergens. One of the more recent reviews of
aptamer-based analysis in context of food safety control indicated
that while a great variety of commercial analytical tools have been
developed for allergen detection, most of them rely on
immunoassays. It was further indicated that the selection of
aptamers for this group of ingredients is emerging (Amaya-Gonzalez
et al., Sensors 2013, 13, 16292-16311, the contents of which are
incorporated herein by reference in their entirety).
[0093] The present disclosure provides detection assemblies and
systems that can specifically detect low concentrations of
allergens in a variety of food samples. The detection system and/or
device of the present disclosure is a miniaturized, portable and
hand-held product, which is intended to have a compact size which
enhances its portability and discreet operation. A user can carry
the detection system and device of the present disclosure and
implement a rapid and real-time test of the presence and/or absence
of one or more allergens in a food sample, prior to consuming the
food. The detection system and device, in accordance with the
present disclosure, can be used by a user at any location, such as
at home or in a restaurant. The detection system and/or device
displays the test result as a standard readout and the detection
can be implemented by any user following the simple instructions on
how to operate the detection system and device. A specific utility
of this detection system is the ease and rapidity of the system.
The detection systems and assemblies of the present disclosure may
also be used to detect any molecule of interest (i.e., any target)
in a sample in general; the molecule of interest may be a protein
or a variant thereof, a nucleic acid molecule (e.g., a DNA or RNA
molecule) or a variant thereof, a lipid, a sugar, a small molecule,
or a cell.
[0094] In some embodiments, the detection system is constructed for
simple, fast, and sensitive one-step execution from the
introduction of the sample to the system. The system may complete a
detection test in less than 10 minutes, less than 9 minutes, less
than 8 minutes, less than 7 minutes, less than 6 minutes, less than
5 minutes, or less than 4 minutes, or less than 3 minutes, or less
than 2 minutes, or less than 1 minute. In some examples, the
detection may be completed in approximately 60 seconds, 55 seconds,
50 seconds, 45 seconds, 40 seconds, 35 seconds, 30 seconds, 25
seconds, 20 seconds, or 15 seconds.
[0095] In accordance with the present disclosure, the detection
system may involve a mechatronic construction process integrating
electrical engineering, mechanical engineering and computing
engineering to implement and control the process of a target
detection test, including but not limiting to, rechargeable or
replaceable batteries, motor drivers for processing the test
sample, pumps for controlling the flow of the processed sample
solutions and buffers within the cartridge, printed circuit boards,
and connectors that couple and integrate different components for a
fast allergen testing. The detection device of the present
disclosure also includes an optical system which is configured for
detection of the presence and concentration of a molecule of
interest (e.g., an allergen) in a test sample and conversion of
detection signals into readable signals; and a housing which
provides support for other parts of the detection device and
integrates different parts together as a functional product.
[0096] In some embodiments, the detection system is constructed
such that disposable analytical cartridges (e.g., a disposable test
cup or cup-like container), unique to one or more specific
molecules of interest (e.g., allergens), are constructed for
receiving and processing a test sample and implementing the
detection test, in which all the solutions are packed. Therefore,
all the solutions may be confined in the disposable analytical
cartridges. As a non-limiting example, a disposable peanut test cup
may be used to detect peanut in any food sample by a user and
discarded after the test. This prevents cross-contamination when
different allergen tests are performed using the same device. In
some embodiments, a separate sampler for collecting a test sample
is provided.
[0097] In accordance with the present disclosure, the disposable
analytical cartridge comprises detection agents that specifically
bind to and recognize an allergen or a molecule of interest. The
detection agents may be, but are not limited to, antibodies or
variants thereof, nucleic acid molecules or variants thereof, and
small molecules. In some embodiments, the detection agents may be
nucleic acid molecules comprising nucleic acid sequences that
specifically bind to a molecule of interest. The nucleic acid-based
detection agents may be aptamers and signaling polynucleotides
(SPNs) derived from aptamers that can recognize the target molecule
such as an allergen. In some embodiments, the SPNs capture the
target molecules in the sample to form SPN:target complexes.
Another detection probe comprising short nucleic acid sequences
that are complementary to the SPN sequence may be used to anchor
the SPN to a solid substrate for signal detection. In other
embodiments, the detection agents and detection probes may be
attached to a solid substrate such as the surface of a magnetic
particle, silica, agarose particles, polystyrene beads, a glass
surface, a plastic chip, a microwell, a chip (e.g., a microchip),
or the like. It is within the scope of the present disclosure that
such detection agents and detection probes can also be integrated
into any suitable detection systems and instruments for similar
purposes.
Detection Assemblies and Systems
[0098] In accordance with the present disclosure, a detection
system or assembly for implementing a detection test of a molecule
of interest (e.g., an allergen) in a sample comprises at least one
disposable analytical cartridge for processing the sample to a
state permitting the molecule of interest to engage in an
interaction with a detection agent, and a detector unit for
detecting and visualizing the result of the detection (i.e., the
interaction between the molecule of interest and the detection
agent). Optionally, the detection system may further comprise at
least one sampler for collecting a test sample. The sampler can be
any tool that can be used to collect a portion of a test sample,
e.g., a spoon. In some aspects, a particularly designed sampler may
be included to the present detection system as discussed
hereinbelow. The exemplary embodiments described below illustrate
such detection systems and assemblies for detecting an allergen in
a sample.
[0099] In general, the analytical cartridge is configured to accept
the sample for processing to a state permitting the molecule of
interest to engage in an interaction with a detection agent. The
detector unit is configured to accept the analytical cartridge in a
configuration which permits a detection mechanism housed by the
detector unit to detect the interaction of the molecule of interest
with the detection agent. The interaction triggers a visual
indication on the detector unit that the molecule of interest is
present or absent in the sample. The detector unit may be removably
connected to the analytical cartridge.
[0100] As shown in FIG. 1, an embodiment of the detection system or
assembly of the present disclosure comprises a detection device 100
configured for processing a test sample, implementing an allergen
detection test, and detecting the result of the detection test, a
separate food corer 200 as an example of the sampler, and a
disposable test cup 300 as an example of the analytical cartridge.
The detection device 100 includes an external housing unit 101 that
provides support to the components of the detection device 100. A
port or receptacle 102 of the detection device 100 is constructed
for docking the disposable test cup 300 and a lid 103 is included
to open and close the instrument. The external housing unit 101
also provides surface space for buttons that a user can operate the
device. An execution/action button 104 that allows a user to
execute an allergen detection testing and a USB port 105 may be
included. Optionally a power plug (not shown) may also be included.
During the process of implementing an allergen detection test, the
food corer 200 with a sample contained therein is inserted into the
disposable test cup 300 and the disposable test cup 300 is inserted
into the port 102 of the detection device 100 for detection.
Sampler
[0101] Collecting an appropriately sized sample is an important
step for implementing allergen detection testing. In some
embodiments of the present disclosure, a separate sampler for
picking up and collecting test samples (e.g. food samples) is
provided. In one aspect, a coring-packer-plunger concept for
picking up and collecting a food sample is disclosed herein. Such
mechanism may measure and collect one or several sized portions of
the test sample and provide pre-processing steps such as cutting,
grinding, abrading and/or blending, for facilitating the
homogenization and extraction or release of allergen proteins from
the test sample. The sampler may be operatively connected to the
analytical cartridge and the detection device for transferring a
test sample to the cartridge for sample processing. According to
the present disclosure, a separate food corer 200 is constructed
for obtaining different types of food samples and collecting an
appropriately sized portion of a test sample. In one example, the
sample is a liquid sample. In another example, the sample is a
solid sample.
[0102] As shown in FIG. 2A, an embodiment of the food corer 200 may
comprise three parts: a plunger 210 at the distal end, a handle 220
configured for coupling a corer 230 at the proximal end. The
plunger 210 has a distal portion provided with a corer top grip 211
(FIG. 2A) at the distal end, which facilitates maneuverability of
the plunger 210 up and down, a plunger stop 212 in the middle of
the plunger body, and a seal 213 at the proximal end of the plunger
body. The handle 220 may comprise a snap fit 221 at the distal end
and a projecting flat collar at the proximal end connecting to the
corer 230. In one embodiment, the projecting flat collar comprises
a flange 222 as shown in FIG. 2A. The corer 230 may comprise a
proximal portion provided with a cutting edge 231 at the very
proximal end (FIG. 2A). The corer 230 is configured for cutting and
holding the collected sample to be expelled into the disposable
test cup 300.
[0103] In some embodiments, the distal end of the plunger 210 may
comprise a push plate. The plate may be a flat plate, in any shape.
In one preferred embodiment, the push plate may be in a rounded
square shape with a flared surface. Additionally, the rounded
square shape provides an anti-roll feature when the sampler 200 is
on a flat surface. This feature also can keep the collected sample
inside the corer 230 (i.e. the sample area) from contacting an
outside surface (e.g. a table when the sampler is lying on the
table).
[0104] In some embodiments, the projecting flat collar may be
configured as a small circular ring, a rib, or the like. This
projection may prevent fingers from sliding down into the sample
area and provide tactile orientation as well. As a non-limiting
example, the projecting flat collar is a small circular ring.
[0105] In one embodiment, the plunger 210 may be inserted inside
the corer 230, where the proximal end of the plunger 210 may
protrude from the corer 230 for directly contacting a test sample,
and together with the cutting edge 231 of the corer 230, picking up
a sized portion of the test sample (FIG. 2B). In accordance with
the present disclosure, the plunger 210 is used to expel sampled
food from the corer 230 into the disposable test cup 300, and to
pull certain foods into the corer 230 as well, such as liquids and
creamy foods. The feature of the plunger stop 212, through an
interaction with the snap fit 221, may prevent the plunger 210 from
being pulled back too far or out of the corer body 230 during
sampling. The seal 213 at the very proximal end of the plunger 210
may maintain an air-tight seal in order to withdraw liquids into
the corer 230 by means of pulling the plunger 210 back. In some
embodiments, the plunger 210 may be provided with other types of
seals including a molded feature, or a mechanical seal. The handle
220 is constructed for a user to hold the coring component of the
sampler 200. For example, the skirt 222 gives the user means for
operating the food sampler 200, pushing down the corer 230 and
driving the corer 230 into the food sample to be collected.
[0106] In some embodiments, the plunger 210 may comprise markings
to provide additional guidance to the user, indicating the position
of the plunger inside the corer and its position relevant to the
minimal and maximal sampling lines. In some embodiments, the lines
indicating the minimal and maximal amounts of the sample to be
collected are added to the exterior of the corer 230. A user can
correct the size of the sampling compartment by adjusting the
minimal and maximal lines.
[0107] In some embodiments, the cutting edge 231 may be configured
for pre-processing the collected sample, allowing the sampled food
to be cored in a pushing, twisting and/or cutting manner. The
cutting edge 231 may cut a portion from the test sample. As some
non-limiting examples, the cutting edge 231 may be in a flat edge,
a sharp edge, a serrated edge with various numbers of teeth, a
sharp serrated edge and a thin wall edge. In other aspects, the
inside diameter of the corer 230 varies, ranging from about 5.5 mm
to 7.5 mm. Preferably the inside diameter of the corer 230 may be
from about 6.0 mm to about 6.5 mm. The inside diameter of the corer
230 may be 6.0 mm, 6.1 mm, 6.2 mm, 6.3 mm, 6.4 mm, 6.5 mm, 6.6 mm,
6.7 mm, 6.8 mm, 6.9 mm, or 7.0 mm. The size of the corer 230 is
optimized for a user to collect a right amount of the test sample
(e.g., 1.0 g to 0.5 g).
[0108] The parts of the food corer 200 may be constructed as any
shape for easy handling such as triangular, square, octagonal,
circular, oval, and the like.
[0109] In some embodiments, the plunger 210 and the other parts of
the sampler may be in different colors. As a non-limiting example,
the plunger may be in green color and the corer may be transparent.
The increased contrast provides a clear view of the position of the
plunger with respect to the sampler. In other embodiments, the food
corer 200 may be further provided with a means for weighing a test
sample being picked up, such as a spring, a scale or the equivalent
thereof. As a non-limiting example, the food corer 200 may be
provided with a weigh tension module.
[0110] The food corer 200 may be made of plastic materials,
including but not limited to, polycarbonate (PC), polystyrene (PS),
poly(methyl methacrylate) (PMMA), polyester (PET), polypropylene
(PP), high density polyethylene (HDPE), polyvinylchloride (PVC),
thermoplastic elastomer (TPE), thermoplastic urethane (TPU), acetal
(POM), polytetrafluoroethylene (PTFE), or any polymer, and
combinations thereof.
[0111] In some embodiments, the sampler may be further configured
to be user friendly. For example, the handle 220 may comprise a
textured surface to create better visual and tactile
differentiation between the grip area and sample areas,
communicating the user where to hold the sampler 200.
[0112] The sampler (e.g., the corer 200) may be operatively
associated with an analytical cartridge (e.g., the disposable cup
300) and/or a detection device (e.g. the device 100). Optionally,
the sampler may comprise an interface for connecting to the
cartridge. Optionally, a cap may be positioned on the proximal end
of the sampler. The sampler 200 may also comprise a sensor
positioned with the sampler 200 to detect a presence of a sample in
the sampler.
Disposable Analytical Cartridge
[0113] In some embodiments, the present disclosure provides an
analytical cartridge or vessel. As used herein, the terms
"cartridge", "vessel" and "test cup" are used interchangeably. The
analytical cartridge is constructed for implementing a detection
test. As used herein, the analytical cartridge is also referred to
as an analytic module. The analytical cartridge is disposable and
used for one particular allergen or a particular set of allergens
(e.g., a set of tree nuts allergens). A disposable analytical
cartridge is constructed for processing a test sample to a state
permitting the allergen(s) of interest to engage in an interaction
with a detection agent, for example, dissociation of food samples
and allergen protein extraction, filtration of food particles,
storage of reaction solutions/reagents and detection agents,
capture of an allergen of interest using detection agents such as
antibodies and nucleic acid molecules that specifically bind to
allergen proteins. In one aspect, the detection agents are nucleic
acid molecules such as aptamers and/or aptamer derived SPNs. In
other embodiments, the detection agents may be antibodies specific
to allergen proteins, such as antibodies specific to peanut
allergen proteins Ara H1. In other embodiments, the detection
agents may be any agents, e.g., chemical compounds, peptide
aptamers and complexes that can specifically recognize allergen
proteins. The present disclosure discusses food allergens as
examples of molecules of interest that can be detected with the
present assemblies. One skilled in the art would understand, any
targets (i.e., molecules of interest) in a sample can be
detected.
[0114] In accordance with the present disclosure, at least one
separate analytical cartridge is provided as part of the assembly.
In other embodiments, the analytical cartridge may be constructed
for use with any other detection systems.
[0115] In some embodiments, a disposable analytical cartridge is
intended to be used only once for an allergen test in a sample and
therefore may be made of low cost plastic materials, for example,
acrylonitrile butadiene styrene (ABS), COC (cyclic olefin
copolymer), COP (cyclo-olefin polymer), transparent high density
polyethylene (HDPE), polycarbonate (PC), poly(methyl methacrylate)
(PMMA), polypropylene (PP), polyvinylchloride (PVC), polystyrene
(PS), polyester (PET), or other thermoplastics. Accordingly, a
disposable analytical cartridge may be constructed for any
particular allergen of interest. In some embodiments, these
disposable cartridges may be constructed for one particular
allergen only, which may avoid cross contamination with other
allergen reactions.
[0116] In some embodiments, the disposable cartridge is made of
polypropylene (PP), COC (cyclic olefin copolymer), COP
(cyclo-olefin polymer), PMMA (poly(methyl methacrylate), or
acrylonitrile butadiene styrene (ABS).
[0117] In other embodiments, these analytical cartridges may be
constructed for detecting two or more different allergens in a test
sample in parallel. In some aspects, the cartridges may be
constructed for detecting two, three, four, five, six, seven, or
eight different allergens in parallel. In one aspect, the presence
of multiple allergens, e.g., two, three, four, five, or more, are
detected simultaneously, a positive signal may be generated
indicating which allergen is present. In another aspect, a system
is provided to detect if an allergen, e.g., peanut or a tree-nut,
is present and generate a signal to indicate the presence of such
allergen.
[0118] In some embodiments, the disposable analytical cartridge may
further be constructed to comprise a bar code that can store the
lot specific parameters. The stored information may be later read
and stored in any digital formats by the user.
[0119] In some embodiments, the analytical cartridge comprises a
homogenizer configured to produce a homogenized sample, thereby
releasing the molecule of interest from a matrix of the sample into
an extraction buffer that optionally includes the detection agent.
The analytical cartridge also comprises a first conduit to transfer
the homogenized sample with or without the detection agent through
a filter system to provide a filtrate containing the molecule of
interest and the detection agent and a second conduit to transfer
the filtrate, making the filtrate to be contacted with a detection
probe, thereby permitting an interaction of the detection agent
with the detection probe. The first and second conduits comprise a
plurality of fluidic paths connecting different parts of the
conduits from transferring the processed sample, buffers, filtrate,
waste and other fluids.
[0120] In some embodiments, the analytical cartridge may further
comprise a rotary valve system providing a mechanism for
controlling the transfer of the sample and other fluidic components
(e.g., buffer, filtrate and waste) in the analytical cartridge. The
rotary valve switching system may be further configured to provide
a closed position to prevent fluid movement in the analytical
cartridge.
[0121] In some embodiments, the homogenizer and the rotary valve
system may be powered by motors located in the detector unit when
the analytical cartridge is accepted by the detector unit, or any
other motor mechanisms provided by a connected detection
device.
[0122] In some embodiments, the analytical cartridge may be
constructed to comprise one or more separate chambers, each
configured for separate functions such as sample reception, protein
extraction, filtration, storage for buffers, agents and waste
solution, and detection reaction. The chambers are separate but
connected for operation. For example, the analytical cartridge may
include a sample processing chamber, a detection chamber, a waste
chamber, and optionally a buffer chamber. In some embodiments, the
analytical cartridge may further comprise a separate filtrate
chamber to hold the filtrate and optionally further concentrate the
filtrate prior to the transfer to the detection chamber. In some
examples, the detection chamber may comprise a detection sensor and
an optical window. The detection mechanism of the detector unit
analyzes the detection reaction through the optical window to
identify the interaction of the molecule of interest with the
detection agent in the detection chamber. The detection window is
operatively associated with the detection mechanism of a detection
device.
[0123] In some embodiments, the analytical cartridge comprises a
detection sensor for measuring the interaction between the target
molecule and the detection agent. The detection sensor is included
in the detection chamber. In one non-limiting example, the
detection sensor is a transparent substrate which includes a
plurality of fluidic channels and a detector chip area. The
substrate is referred to as a chipannel, wherein the fluidic
channels and the detector chip area are connected. In some
examples, the chipannel is a plastic substrate.
[0124] In some embodiments, the detector chip area within the
chipannel comprises at least one reaction panel and at least one
control panel. In other embodiments, the detector chip area within
the chipannel may comprise one reaction panel and two control
panels. In other embodiments, the chipannel may comprise a
plurality of reaction panels and a plurality of control panels.
Optionally, the detector chip area of the chipannel further
comprises one or more fiducial spots that guide image processing by
an imaging mechanism (e.g., a camera) of the detector unit. Any
suitable fiducial object may be spotted as a fiducial marker for
reference.
[0125] In some embodiments, the chipannel comprises a detection
probe molecule immobilized on the reaction panel of the detector
chip area. The detection probe is configured to engage in a probe
interaction with the detection agent. An interaction of the
molecule of interest with the detection agent prevents the
detection agent from engaging in the probe interaction with the
detection probe. The detector chip area within the chipannel may
further include an optically detectable control probe molecule
immobilized on the control panel(s), for normalization of signal
output measured by the detection mechanism. In some embodiments,
the control probe molecule is a nucleic acid molecule that does not
bind to the molecule of interest or the detection agent.
[0126] In one preferred embodiment, the chipannel is a plastic chip
wherein the reaction panel is printed with a nucleic acid-based
detection probe that comprises a nucleic acid sequence
complementary to nucleic acid sequence of the detection agent and
wherein the control panel is printed with nucleic acid based
control probe molecule that does not bind to the detection
agent.
[0127] In some embodiments, detection agents, detection probes,
buffers such as extraction buffers and wash buffers, and other
components necessary for assembling a functional cartridge are
included.
[0128] In some embodiments, the analytical cartridge may comprise a
data chip unit configured for providing the cartridge
information.
[0129] In accordance with the present disclosure, the analytical
cartridge may be construed in any suitable shape and size. Some
exemplary embodiments of the analytical cartridge are illustrated
below. The exemplary embodiments do not intent to limit the design
of the cartridge.
Exemplary Embodiments of the Analytical Cartridge
[0130] In some embodiments, the disposable analytical cartridge may
be construed as a disposable test cup or a cup-like container. The
cup container may comprise several compartments that are assembled
into a functional analytic module. According to one embodiment of
the test cup, as shown in FIG. 3A, the assembled disposable test
cup 300 comprises three parts: a cup top 310, a cup body 320 and a
cup bottom 330. The three parts are operatively connected to
assemble a functional analytical module. The cup 300 further
comprises a homogenization rotor 340 that rotates in both
directions to homogenize the sample, a filter assembly 325
filtrating the processed sample, a rotary valve 350 contemplated to
control the fluid flow inside the cup (FIG. 3B), and fluidic paths
transporting the processed sample, mixer, filtrate, buffers and
agents to different compartments of the test cup (not shown in FIG.
3B).
[0131] The test cup body 320 may include a plurality of chambers.
In one embodiment, as shown in FIG. 3B, the test cup body 320
includes one homogenization chamber 321 comprising a food
processing reservoir 801 (as shown in FIG. 8C), a filtrate chamber
322 for collecting a sample solution after being filtered through
the filter (e.g., the 2-state filter 325 shown in FIG. 3B and FIG.
4A), a waste chamber 323 comprising a waste reservoir 803 (as shown
in FIG. 8C), and optionally, a wash buffer storage chamber 324
comprising wash buffer storage reservoir 802 (as shown in FIG. 8C).
Optionally, one or more separate wash compartments may be included
in the cup body 320. In some embodiments, a reaction chamber 331 at
the cup bottom 330 for receiving the processed sample (also
referred to herein as a signal detection chamber) is included shown
in FIGS. 3B and 3H. The reaction/detection chamber 331 may comprise
a separate detection sensor (e.g., the chip 333 shown in FIG. 3B)
with a detection probe that reacts with the processed sample. All
analytical reactions occur in the reaction/detection chamber 331,
and a detectable signal (e.g., a fluorescence signal) is generated
therein. In some embodiments, detection agents (e.g., SPNs) for
example, which are pre-stored in the homogenization chamber 321,
may be premixed with the test sample in the homogenization chamber
321, where the test sample is homogenized and the extracted
allergen proteins react with the detection agents. The mixed
reaction complexes may be transported to the filter 325 before they
are transported to the reaction/detection chamber 331. In other
examples, detection agents (e.g., SPNs) may be stored in the
filtrate chamber 322. The processed sample is filtered through the
filter assembly 325 and reacts with the detection agents stored in
the filtrate chamber 322. The filtrate containing the molecule of
interest and detection agents is transferred to the detection
chamber 331 wherein the detection agents engage an interaction with
the detection probes immobilized on the sensor (e.g., the chip 333)
and the detection signal is measured.
[0132] In alternative embodiments, more than one buffer and reagent
storage reservoir may be included in the buffer and reagent storage
chamber 324. As a non-limiting example, the extraction buffer and
wash buffers may be stored separately in a reservoir within the
buffer storage chamber 324.
[0133] FIG. 3C shows an exploded view of one exemplary embodiment
of the disposable test cup 300 which is configured to contain three
main components, the top 310, the housing or body 320 and the
bottom 330. The cup top 310 may include a cup lid 311, a top cover
312, two or more breather filters 314 which are included to ensure
that only air is brought in and that fluids do not escape from the
test cup 300. The cup body 320 is composed of two separate parts:
an upper housing 320a and an outer housing 320b. The cup bottom
assembly 330 includes a bottom cover 337 that sandwiches other
components including the reaction chamber 331 (in FIGS. 3F and 3H),
a detection sensor, i.e. a glass chip 333, and a chip gasket 334
that facilitates the attachment of the glass chip 333 to the bottom
of the specialized sensor area 332 in the reaction chamber 331. In
some embodiments, the processed sample mixer flows to the reaction
chamber 331 and reacts with the detection agents on the chip 333 to
generate detectable signals. For example, the chip 333 may be
coated with oligonucleotide sequences to detect targets presented
in the test sample. The bottom cover 337 also comprise a port/bit
340a for holding the homogenization rotor 340 and a port/bit 350a
for holding the rotary valve 350 (as shown in FIG. 3H). These bits
provide a means for linking the homogenization rotor 340 and the
rotary valve 350 to the motors of the detection device 100. In some
embodiments, a rotor gasket 326 may be configured to the upper
housing 320a to seal the rotor 340 to the housing 320, to avoid
leakage of fluids. In some embodiments, the bottom cover may
further comprise fluidic paths and air channels.
[0134] In some embodiments, the cup may further be constructed to
comprise a bar code that can store the lot specific parameters. In
one example, the bar code may be the data chip 335 that stores the
cup 300 specific parameters, including the information of detection
agents such as SPNs (e.g., fluorophore labels, the target allergen,
and intensity of SPNs, etc.), expiration date, manufacture
information, etc.
[0135] FIG. 3D further demonstrates the features of the top cover
312 of the cup shown in FIG. 3A. A corer port 313 is included for
receiving a food corer 200, thereby receiving the picked test
sample and transferring the sample to the sample processing chamber
321 (also referred to as homogenization chamber). As a non-limiting
example, the port 313 may be configured for receiving the food
corer 200 shown in FIG. 2B. The top cover 312 may also include at
least one small hole (FIG. 3D) for air to be drawn in for fluid
flow. As a non-limiting example, the top part may have two lids
311. As discussed hereinabove, the lid 311 may comprise two layers:
a top lid 311a for sealing and labeling and a bottom 311b for
resealing during operation. As shown in FIG. 3E, the second lid at
the bottom 311b is constructed for resealing and liquid retention
during the operation. The top lid 311a may be peeled back for
inserting the test sample collected by the corer 200, and then
reclosed after assay completion.
[0136] FIG. 3F is a top view of a cup housing body 320 as the upper
housing 320a and the outer housing 320b are assembled together
(left panel). The upper housing 320a may comprise one or more
chambers which are operatively connected. In one embodiment, the
homogenization chamber 321, filtration chamber 322 and waste
chamber 323 are included in the housing 320a (left panel). Two
breath filters 314 are also added to the upper housing 320a. The
bottom of the assembled cup body 320 comprises an opening 331a that
connects to the reaction/detection chamber 331 with the inlet and
outlet 336 for fluid flow (right panel). In this embodiment, the
reaction/detection chamber 331 is formed when the bottom cover 337
is assembled together with the body part (see FIG. 3C) The rotor
340 and the rotary valve 350 may be assembled into the cup to form
an analytical cartridge (right panel).
[0137] FIG. 3G further illustrates the outer interface of the
bottom of the upper housing (320a) (upper panel) and the inner
interface of the bottom of the outer housing 320b (lower panel).
The two energy director faces 361 (face 1) and 362 (face 2) at the
outer interface of the upper housing 320a, interact with the two
welding mating faces, face 363 (face 1) and 364 (face 2) at the
inner interface of the bottom of the outer housing 320b to retain
the housing parts 320a and 320b together to form the cup body 320.
Fluid paths 370 are also included to flow liquids to the cup bottom
330. The rotor 340 and the rotary valve 350 are assembled into the
cup through the rotor port 340a and the rotary valve port 350a,
respectively.
[0138] FIG. 3H further illustrates the cup bottom cover 337 of the
cup bottom 330 of the cup 300 shown in FIG. 3A and FIG. 3C. The
reaction/detection chamber 331 comprises a specialized sensor area
332 where a detection sensor, i.e. the glass chip 333, is
positioned through a glass gasket 334. The glass gasket 334 may be
included to seal the glass chip 333 in place to the bottom of the
reaction chamber 331 and to prevent fluid leakage. Alternatively,
adhesive or ultrasonic bonding can be used to mate the layers
together. In some aspects of the present disclosure, the glass chip
333 may be configured directly at the bottom of the reaction
chamber 331 (e.g., the bottom surface of the sensor area 332) as a
component of the cup bottom cover 337. and integrated into the cup
body as one entity. The entire unit may be of PMMA (poly(methyl
methacrylate)) (also referred to as acrylic or acrylic glass). This
transparent PMMA acrylic glass may be used as optic window for
signal detection.
[0139] The reaction chamber 331 comprises at least one optical
window. In one embodiment, the chamber 331 comprises two optical
windows, one primary optical window and one secondary optical
window. In some embodiments, the primary optical window serves as
the interface of the reaction chamber 331 to the detection device
100, in particular to the optical system 1030 (as shown in FIGS.
10A, 10B, and 12A-12C) of the detection device 100. The detection
sensor (e.g., the glass chip 333) may be positioned between the
optical window and the interface of the optical system. The
optional secondary optical window may locate at one side of the
reaction chamber 331; the secondary optical window allows detection
of the background signals. In some aspects of the present
disclosure, the secondary optical window may be constructed for
measuring scattered light.
[0140] As shown in FIG. 3I, the bottom 330 is assembled together
with the cup body 320. From this bottom perspective view, the
bottom surface comprises several interfaces for fluid paths (e.g.
fluidic inlet/outlet 336) and a plump interface 380 and the
interfaces connecting the rotor 340 and the rotary valve 350 to the
detection device 100.
[0141] A means may be included to the cup to block the fluid flows
between the compartments of the assembled cup 300. In one
embodiment, a dump valve 315 (shown in FIG. 3C) in the cup housing
320a is included to block fluid in the homogenization chamber 321
from flowing to the rotary valve 350 that is configured at the
bottom of the cup 300. The dump valve 315 is held in place by the
rotary valve 350 (FIG. 3C) for shipping, storage and end of life.
The rotary valve 350 locks the dump valve 315 over the filters
(e.g., the filter assembly 325) during shipping and prevents fluid
flow after completing the detection assay. The rotary valve 350 may
be actuated in several steps to direct fluid flow to the proper
chambers. As a non-limiting example, the relevant positions of the
rotary valve 350 during the detection test are demonstrated in FIG.
8B.
[0142] The rotary valve 350 may rotate to regulate the fluid flow
through the chambers inside the cartridge. In some embodiments, the
rotary valve 350 may comprise a valve shaft 351 that is operatively
connected to and locks the dump valve 315 (as shown in FIG. 3C) and
a valve disc 352 connected to the valve shaft 351 (e.g., in FIG.
6F). The rotary valve 350 can be attached to the cup through any
available means known in the art. In one embodiment, a valve gasket
(e.g., the gasket 504 shown in FIG. 5B) may be used. Alternatively,
the rotary valve can be attached to the cup through a disc spring
(e.g., a wave disc spring). In another embodiment, the rotary valve
350 may be secured to the cup with a plurality of compression coil
springs (e.g., 721 shown in FIG. 7J).
[0143] In some embodiments, a filter assembly (e.g., the filter 325
shown in FIG. 3C, FIG. 4A and FIG. 6D) is included in the
analytical cartridge. The filter removes large particles and other
interfering components from the sample, such as fat from a food
matrix, before the processed sample is transferred into the
reaction chamber 331.
[0144] In some embodiments, the filter mechanism may be a filter
assembly. The filter assembly may be a simple membrane filter 420.
The membrane 420 may be a nylon, PE, PET, PES (poly-ethersulfone),
Porex.TM., glass fiber, or the membrane polymers such as mixed
cellulose esters (MCE), cellulose acetate, PTFE, polycarbonate,
PCTE (Polycarbonate) or PVDF (polyvinylidene difluoride), or the
like. It may be a thin membrane (e.g., 150 .mu.m thick) with high
porosity. In some aspects, the pore size of the filter membrane 420
may range from 0.01 .mu.m to 600 .mu.m, or from 0.1 .mu.m to 100
.mu.m, or from 0.1 .mu.m to 50 .mu.m, or from 1 .mu.m to 20 .mu.m,
or from 20 .mu.m to 100 .mu.m, or from 20 .mu.m to 300 .mu.m, or
100 .mu.m to 600 .mu.m or any size in between. For example, the
pore size may be about 0.02 .mu.m, about 0.05 .mu.m, about 0.1
.mu.m, about 0.2 .mu.m, about 0.5 .mu.m, about 1.0 .mu.m, about 1.5
.mu.m, about 2.0 .mu.m, about 2.5 .mu.m, about 3 .mu.m, about 3.5
.mu.m, about 4.0 .mu.m, about 4.5 .mu.m, about 5.0 .mu.m, about 10
.mu.m, about 15 .mu.m, about 20 .mu.m, about 25 .mu.m, about 30
.mu.m, about 35 .mu.m, about 40 .mu.m, about 45 .mu.m, about 50
.mu.m, about 55 .mu.m, about 60 .mu.m, about 65 .mu.m, about 70
.mu.m, about 75 .mu.m, about 80 .mu.m, about 85 .mu.m, about 90
.mu.m, about 100 .mu.m, about 150 .mu.m, about 200 .mu.m, about 250
.mu.m, about 300 .mu.m, about 350 .mu.m, about 400 .mu.m, about 450
.mu.m, about 500 .mu.m, about 550 .mu.m, or about 600 .mu.m.
[0145] In some alternative embodiments, the filter assembly may be
a complex filter assembly 325 (as shown in FIG. 4A) comprising
several layers of filter materials. In one example, the filter
assembly 325 may comprise a bulk filter 410 composed of a gross
filter 411, a depth filter 412, and a membrane filter 420 (FIG.
4A). In one embodiment, the gross filter 411 and the depth filter
412 may be held by a retainer ring 413 to form a bulk filter 410
sitting on the membrane filter 420. In other embodiments, the bulk
filter 410 may further comprise a powder that sits inside the
filter or on top of the filter. The powder may be selected from
cellulose, PVPP, resin, or the like. In some examples, the powder
does not bind to nucleic acids and proteins.
[0146] In some embodiments, the filter assembly 325 may be
optimized for removing oils from highly fatty samples, but not
proteins and nucleic acids, resulting in superior sample cleaning.
In other embodiments, the ratio of the depth and width of the
filter assembly 325 may be optimized to maximize the filtration
efficiency.
[0147] In some embodiments, the filter assembly 325 may be placed
inside a filter bed chamber 431 in the disposable cup body 320. The
filter bed chamber 431 may be connected to the homogenization
chamber 321. The homogenate can be fed to the filter assembly 325
inside the filter bed chamber 431. The filtrate is collected by the
collection gutter 432 (also referred to herein as filtrate chamber)
(FIG. 4B). The collected filtrate then can exit the fluidics to
flow to the reaction chamber 331 (FIG. 3B). In one example, the
collected filtrate may be transported to the reaction chamber 331
from the collection gutter 432 directly. In another example, the
filtrate may be first transported to the filtrate collection
chamber 433 before being transported to the reaction chamber 331
through the inlet/outlet 336 (FIG. 3H). The fluids may be delivered
to the reaction chamber 331 by the fluid paths 370 at the bottom of
the cup 320 (as shown in FIG. 3G).
[0148] In some embodiments, the filtrate collection chamber 433 may
further comprise a filtrate concentrator which is configured to
concentrate the sample filtrate before it flows to the reaction
chamber 331 for signal detection. The concentrator may be in a
half-ball shape, or a conical type concentrator, or a tall
pipe.
[0149] In accordance, the processed sample (e.g., the homogenate
from the chamber 321) is filtered sequentially through the gross
filter 411, the depth filter 412 and the membrane filter 420. The
gross filter 411 can filter a large particle suspension from the
sample, for example, particles larger than 1 mm, and/or some dyes.
The depth filter 412 may remove small particle collections and oil
components from the sample (such as the food sample). The pore size
of the depth filter 412 may range from about 1 .mu.m to about 500
.mu.m, or about 1 .mu.m to about 100 .mu.m, or about 1 .mu.m to
about 50 .mu.m, or about 1 .mu.m to about 20 .mu.m, or about 4
.mu.m to about 20 .mu.m, or from about 4 .mu.m to about 15 .mu.m.
For example, the pore size of the depth filter 412 may be about 2
.mu.m, or about 3 .mu.m, or about 4 .mu.m, or about 5 .mu.m, or
about 6 .mu.m, or about 7 .mu.m, or about 8 .mu.m, or about 9
.mu.m, or about 10 .mu.m, or about 11 .mu.m, or about 12 .mu.m, or
about 13 .mu.m, or about 14 .mu.m, or about 15 .mu.m, or about 16
.mu.m, or about 17 .mu.m, or about 18 .mu.m, or about 19 .mu.m, or
about 20 .mu.m, or about 25 .mu.m, or about 30 .mu.m, or about 35
.mu.m, or about 40 .mu.m, or about 45 .mu.m, or about 50 .mu.m.
[0150] The depth filter 412 may be composed of, for example, cotton
including, but not limited to raw cotton and bleached cotton,
polyester mesh (monofilament polyester fiber) and sand (silica). In
some embodiments, the filter material may be hydrophobic,
hydrophilic or oleophobic. In some examples, the material does not
bind to nucleic acids and proteins. In one embodiment, the depth
filter is a cotton depth filter. The cotton depth filter may vary
in sizes. For example, the cotton depth filter may have a ratio of
width and height ranging from about 1:5 to about 1:20. The cotton
depth filter 412 may be configured to correlate total filter volume
and the food mass being filtered.
[0151] The membrane filter 420 can remove small particles less than
10 .mu.m in size, or less than 5 .mu.m in size, or less than 1
.mu.m in size. The pore size of the membrane may range from about
0.001 .mu.m to about 20 .mu.m, or from 0.01 .mu.m to about 10
.mu.m. Preferably the pore size of the filter membrane may be about
0.001 .mu.m, or about 0.01, or about 0.015 .mu.m, or about 0.02
.mu.m, or about 0.025 .mu.m, or about 0.03 .mu.m, or about 0.035
.mu.m, or about 0.04 .mu.m, or about 0.045 .mu.m, or about 0.05
.mu.m, or about 0.055 .mu.m, or about 0.06 .mu.m, or about 0.065
.mu.m, or about 0.07 .mu.m, or about 0.075 .mu.m, or about 0.08
.mu.m, or about 0.085 .mu.m, or about 0.09 .mu.m, or about 0.095
.mu.m, or about 0.1 .mu.m, or about 0.15 .mu.m, or about 0.2 .mu.m,
or about 0.2 .mu.m, or about 0.25 .mu.m, or about 0.3 .mu.m, or
about 0.35 .mu.m, or about 0.4 .mu.m, or about 0.45 .mu.m, or about
0.5 .mu.m, or about 0.55 .mu.m, or about 0.6 .mu.m, or about 0.65
.mu.m, or about 0.7 .mu.m, or about 0.75 .mu.m, or about 0.8 .mu.m,
or about 0.85 .mu.m, or about 0.9 .mu.m, or about 1.0 .mu.m, or
about 1.5 .mu.m, or about 2.0 .mu.m, or about 3.0 .mu.m, or about
3.5 .mu.m, or about 4.0 .mu.m, or about 4.5 .mu.m, or about 5.0
.mu.m, or about 6.0 .mu.m, or about 7.0 .mu.m, or about 8.0 .mu.m,
or about 9.0 .mu.m, or about 10 .mu.m. As discussed above, the
membrane may be a nylon membrane, PE, PET, a PES
(poly-ethersulfone) membrane, a glass fiber membrane, a polymer
membrane such as mixed cellulose esters (MCE) membrane, cellulose
acetate membrane, cellulose nitrate membrane, PTFE membrane,
polycarbonate membrane, Track-Etched polycarbonate membrane, PCTE
(Polycarbonate) membrane, polypropylene membrane, PVDF
(polyvinylidene difluoride) membrane, or nylon and polyamide
membrane.
[0152] In one embodiment, the membrane filter is a PET membrane
filter with 1 .mu.m pore size. The small pore size can prevent
particles larger than 1 .mu.m to pass into the reaction chamber. In
another embodiment, the filter assembly may comprise a cotton
filter combined with a PET mesh having 1 .mu.m pore size.
[0153] In other embodiments, the filter components may be assembled
together by any known methods in the art, such as by heat welding,
ultrasonic welding or a similar process that ensures the assembled
materials can be die-cut and packaged without damaging or
inhibiting the performance of each filter independently or as an
integrated filter assembly. In other embodiments, the packaging of
each part the filter assembly enables high-speed automation systems
on a manufacturing assembly line (e.g., a robotic assembly
line).
[0154] In some embodiments, the filtration mechanism has low
protein binding, low or no nucleic acid binding. The filter may act
as a bulk filter to remove fat and emulsifiers and large particles,
resulting in a filtrate with comparable viscosity to the
buffer.
[0155] In some embodiments, the filter assembly 325 including the
gross filter 411, the depth filter 412 and the membrane filter 420
can allow the maximal recovery of signaling polynucleotides (SPNs)
and other detection agents.
[0156] In other embodiments, the filtration assembly 325 may be
configured to comprise a filter 624 (e. g., a mesh filter) that is
inserted to a filter gasket 623, a bulk filter 622 composed of a
gross filter and a depth filter and a filter cap 621 (as shown in
FIG. 6D). In an alternative embodiment, the filter gasket 623 can
be molded into the cup body as an overmolded component of the cup
body 320, e.g., in the homogenization chamber 321 (as shown in
FIGS. 7E and 7F). The filter 624, the bulk filter 622 and the
filter cap 621 are inserted to the overmolded gasket to form a
functional filter assembly 325.
[0157] In some embodiments, the filtration mechanism can complete
the filtering process in less than 1 minute, preferably in about 30
seconds. In one example, the filtration mechanism may be able to
collect the sample within 35 seconds, or 30 seconds, or 25 seconds,
or 20 seconds with less than 10 psi pressure. In some embodiments,
the pressure may be less than 9 psi, or less than 8 psi, or less
than 7 psi, or less than 6 psi, or less than 5 psi.
[0158] In some alternative embodiments, the filtration chamber 322
may comprise one or more additional chambers conjured for filtering
the processed sample. As illustrated in FIG. 4B, the filtration
chamber 322 may further comprise a separate filter bed chamber 431
wherein a filter assembly 325 (as illustrated in FIG. 4A) is
inserted and connected to a collection gutter 432. The collection
gutter 432 is configured to collect the filtrate that runs through
the filter assembly 325, and the gutter 432 may be directly
connected to the flow cell fluidics to flow the filtrate to the
reaction chamber 331 for signal detection. Optionally, another
collection/concentration chamber 433 may be included in the
filtration chamber 322 which is configured for collecting and/or
concentrating the filtrate collected through the collection gutter
432 before the filtrate is transported to the reaction chamber 331
for signal detection. The collection/concentration chamber 433 is
collected to the filter bed chamber 431 through the collection
gutter 432.
[0159] FIGS. 5A to 5C illustrate another embodiment of the
analytical cartridge. FIG. 5A illustrates an alternative assembly
of the test cup 300. The components of the cup 300 of this
embodiment are shown in FIG. 5B. According to this embodiment, the
cup 300 comprises three parts, a cup top including a cup top cover
310, a cup body comprising a cup tank 320, and a cup bottom
including a cup bottom cover 330, which are operatively connected
to form an analytic module. As illustrated in FIG. 5B, the top of
the cup is a top cover 310 for sealing the cup where a test sample
is placed into the cup for testing. A top gasket 501 may be
included to seal the top 310 to the cup body 320. The upper cup
body 320 comprises the homogenization chamber, waste chamber,
chambers for wash buffers (e.g., wash 1 chamber (W1), wash 2
chamber (W2) (shown in FIG. 6B, right panel), and air vent stacks
for controlling air and thus fluid flow. A rotor 340 is configured
in the homogenization chamber for homogenizing the test sample in
an extraction buffer. The shape of the rotor may be adjusted to fit
the cup during the assembly. A mid gasket 502 is located at the
bottom of the upper cup body 320 to seal the body 320 to the
manifold 520 with holes for fluid flow. The manifold 520 is
configured to hold the filter 325 and the fluid paths 370 for fluid
flow. Another mid gasket 503 is added to seal the manifold 520 to
the cup bottom 330, where the reaction chamber (e.g., chamber 331),
the detection sensor (e.g., glass chip 333), glass gasket (e.g.,
gasket 334) and the memory chip (e.g. EPROM) are located. The rotor
340 is sealed to the bottom through an O-ring 505 (shown in FIG.
5C). The rotary valve 350 is configured to the cup 300 at the
bottom 330 through a valve gasket 504. In another embodiment, the
rotary valve 350 can be configured to the cup 300 through a spring
arm, such as wave disc springs and compression coil springs at the
cup bottom 330 (e.g., 721 shown in FIG. 7J). The configuration of
each components of the cup in FIG. 5A is also illustrated in a
section view in FIG. 5C.
[0160] According to the present disclosure, a third embodiment of
the disposable cup 300 is illustrated in FIG. 6A. FIGS. 6B-6G
further illustrate the components of the disposable cup 300 in FIG.
6A. In this embodiment, the configurations of the detection sensor
and fluidic paths are further integrated. As shown in FIG. 6A, the
cartridge comprises a top part 310, a body part 320 and a bottom
part 330. The rotor 340 is sealed to the cup body 320 through a
gasket 612. The rotary valve 350 is assembled to the cartridge
through a disc spring 613, or alternatively through compression
coil springs at the cup bottom part 330 (e.g., 721 shown in FIG.
7J). When implementing a detection assay, the rotary valve 350 may
rotate and move the seal 612 to free the rotor 340 for homogenizing
the test sample. In this embodiment, a separate panel 631 is
provided between the bottom of the cup body 320 and the bottom
cover 337 in which the fluidic channels are included. This separate
panel 631 with fluidic channels functions equivalently as the
fluidic paths 370 of the previous cup embodiments (e.g., FIGS. 3C,
3G and 3I). The sensor chip 333 may be operatively connected to the
fluidic panel 631 and the sensor area 332 of the reaction chamber
331 in the bottom cover 337 through a chip PSA 632. In an
alternative embodiment, the sensor chip 333 and the fluidic panel
631 may be combined to form a single thin panel (also referred to
as a chipannel), therefore forming a separate chipannel 710 (as
shown in FIG. 7A). The chipannel 710 is discussed in detail
below.
[0161] The cup top 310 may comprise a top lid 311 having two labels
311a and 311b as shown in FIG. 3E, and a top cover 312 as shown in
FIG. 3D. The cup body 320 may be configured for comprising several
separate chambers, including a homogenization chamber 321, a
filtration chamber 322, a waste chamber 323, two or more washing
spaces (W1 and W2) as shown in FIG. 6B (right panel). In some
examples, the filtration chamber 322 has a vent 611 (shown in FIG.
6A). The wetting of the vent 611 can signal to the pressure sensor
of the electronics that the chamber 322 is full (FIG. 6B). Similar
to other designs, at the bottom of the cup body 320 (FIG. 6B, left
panel), several ports are designed including a port 340a for the
rotor 340 and a port 350a for the rotary valve 350 (e.g., the
rotary valve 350 shown in FIG. 6F) for assembling a functional
cartridge. When the cup bottom cover 337 is sealed to the cup body
320 and seals the cup to form a analytic module, these ports are
aligned with the ports of the bottom cover 337 (e.g., 340a and 350a
as shown in FIG. 6C). The sensor chip 333 is attached to the bottom
of the cup body 320 through the chip PSA 632 (FIG. 6B, left
panel).
[0162] FIG. 6C shows a bottom perspective view of the cup bottom
cover 337 and the bottom of the cup body 320 in alignment with each
other, indicating the position of each component upon assembly of
the test cup. When the bottom cover 337 and the cup body 320 are
assembled together, a detection chamber with an optical window
(331) is formed wherein a sensor area 332 holds the sensor chip
333. The optical window of the detection chamber 331 provides a
connection to the detector unit (e.g., the detection device 100 in
FIGS. 1 and 9A).
[0163] In this embodiment, the fluidic panel 631 is positioned
between the bottom of the cup body 320 and the bottom cover 337
(FIG. 6A); the fluidic panel 631 may be operatively connected to a
detection sensor. As a non-limiting example, the fluidic panel 631
is connected to the sensor chip 333 through the chip PSA 632 and
provides essential fluid paths (e.g., 370) for flowing the
processed sample to the detection chamber 331, thereby to the
sensor chip 333.
[0164] In some examples, a filter assembly 325 is inserted to the
homogenization chamber 321 to filtrate the processed sample. In one
example, the filter assembly 325 may be the filter illustrated in
FIG. 4A. In another example, the filter assembly 325 may be
configured to comprise a filter 624 (e. g., a mesh filter) that is
inserted to a filter gasket 623, a bulk filter 622 and a filter cap
621 (FIG. 6D). The filter assembly 325 may be fastened and
controlled by the rotary valve 350 (FIG. 6E). In this embodiment,
the filter cap 621 is engaged in an interaction with the threaded
top of the rotary valve shaft 351 (FIG. 6E). The rotary valve 350
comprises a valve shaft 351 that is operatively connected to and
locks the filter cap 621, a valve disc 352 connected to the valve
shaft 351 (e.g., in FIG. 6F). The valve disc 352 is connected to a
motor of the detector unit upon assembling the test cup to the
detector unit.
[0165] FIG. 6G shows a bottom perspective view (upper panel) and a
top perspective view (lower panel) of the cup bottom cover 337. The
exterior of the bottom cover 337 holds ports (e.g., 340a and 350a)
and the optical window of the sensor area 332 for connecting to the
detection device 100. The interior of the bottom cover 337 includes
the disc spring 613 to secure the rotary valve 350.
[0166] In some embodiments, the reaction chamber 331 at the cup
bottom cover 337 may comprise a specialized sensor area 332 which
is configured for holding a detection sensor for signal detection.
In some aspects of the disclosure, the detection sensor may be a
solid substrate (e.g., a glass surface, a chip, and a microwell) of
which the surface is coated with detection probes such as short
nucleic acid sequences complementary to the SPNs that bind to the
target allergen. In some examples, the detection sensor held at the
sensing area 332 within the reaction chamber 331 may be a glass
chip 333 (as shown in FIGS. 3C and 6A).
[0167] In other embodiments, the reaction chamber 331 comprises at
least one optical window. In one embodiment, the chamber comprises
two optical windows, one primary optical window and one secondary
optical window. Similar to the other embodiments, the primary
optical window serves as the interface of the reaction chamber 331
to the detection device 100, in particular to the optical system
1030 (as shown in FIGS. 10A, 10B, and 12A-12C) of the detection
device 100. The detection sensor (e.g., the glass chip 333, and the
detection area 333' of the chipannel 710) may be positioned between
the optical window and the interface of the optical system. The
optional secondary optical window may locate at one side of the
reaction chamber 331; the secondary optical window allows detection
of the background signals. In some aspects of the present
disclosure, the secondary optical window may be constructed for
measuring scattered light.
[0168] In some embodiments, the glass chip 333 and/or the detection
area 333' of a chipannel 710 that is printed with nucleic acid
molecules (i.e., a DNA chip) is aligned with the optical window. In
some embodiments, the DNA chip comprises at least one reaction
panel and at least one control panel. In some aspects, the reaction
panel of the chip faces the reaction chamber 331, which is flanked
by an inlet and outlet channel 336 of the cartridge 300 (e.g.,
shown in FIGS. 3H and 3I). In some embodiments, the reaction panel
of the glass chip 333 may be coated/printed with detection probes
such as short nucleic acid probes that hybridize to a SPN having
high specificity and binding affinity to an allergen of interest.
The SPN then can be anchored to the chip upon hybridization with
the nucleic acid probes.
[0169] In one preferred embodiment, the sensor DNA chip (e.g., 333
in FIG. 3C, FIG. 5B and FIG. 6A, and 333' in FIG. 7B) may comprise
a reaction panel printed with detection probes comprising short
complementary sequences that hybridize to a SPN specific to an
allergen of interest, and two or more control areas (control
panels) that are covalently-linked to nucleic acid molecules (as
control probes) that do not react with the SPN or the allergen. The
complementary probe sequences can only bind to the SPN when the SPN
is free from binding of the target allergen proteins. In some
aspects, the nucleic acid molecules printed in the control panels
are labeled with a probe, for example, a fluorophore. The control
panels provide an optical set-up with a mechanism to normalize
signal output with respect to the reaction panel and to confirm
functioning operational procedures. An exemplary configuration of
the chip 333 or the detection area 333' is illustrated in FIG.
13A.
[0170] In another embodiment, the sensor DNA chip (e.g., 333 in
FIG. 3C, FIG. 5B and FIG. 6A, and 333' in FIG. 7B) may comprise one
reaction panel printed with detection probes comprising short
complementary sequences that hybridize to a SPN specific to an
allergen of interest, one control area (control panel) that is
covalently-linked to control nucleic acid molecules and one or more
fiducial spots that can guide image processing and provide a
self-correction mechanism for an image detector (e.g., a camera
detector in FIG. 15A). An exemplary configuration of the chip 333
or the detection area 333' is illustrated in FIG. 13C.
[0171] In some embodiments, the DNA coated chip may be pre-packed
into the reaction chamber 331 of the cartridge, e.g., at the
sensing area 332. In other embodiments, the DNA coated chip may be
packed separately with the disposable cartridge (e.g. the cup 300
in FIG. 1). In other embodiments, the DNA chip 333 may be attached
to the fluidic panel 631 shown in FIG. 6A. In other embodiments,
the DNA chip may be integrated to the chipannel as a specialized
detection area of the chipannel (e.g., 333' of the chipannel 710
shown in FIG. 7B).
[0172] Another alternative embodiment of the analytical cartridge
is provided in the present disclosure. The configuration of the
test cup of this alternative the embodiment is shown in FIG. 7A, in
which the test cup 300 comprises a similar configuration of the
compartments (e.g., shown in FIG. 6A) including a cup top 310, a
cup body 320 that is configured to include a homogenization
chamber, a filtrate chamber, wash chambers and a waste chamber, and
a cup bottom 330. This design is simple and requires fewer
components. In this embodiment, a chipannel 710 that combines the
fluidic panel 631, the chip 333 and the chip PSA 632 into a single
thin piece is provided to replace these components. The chipannel
710 may be connected to the cup body 320 through a gasket 701 (FIG.
7A) and the bottom cover 337 via a port connection 711 (FIG. 7C).
Alternatively, the chipannel 710 may be welded to the cup body by a
seal face 712 (e.g., in the alternative embodiment shown in FIG.
7D).
[0173] In some embodiments, the chipannel 710 comprises the fluidic
paths and the sensor chip with detection probes immobilized
thereon, which is made of a separate thin plastic polymer.
According to the present disclosure, the chipannel 710 may be a
piece of plastics in which a specific area (FIG. 7B) is
configurated as the detection area 333' (i.e., an equivalent of the
separate DNA chip 333 in other embodiments). The chipannel 710 may
comprise the fluidic channels (e.g., the paths 370 in FIG. 7B)
connected to the detection area 333'. The detection area 333' may
be flanked by an inlet and outlet channel 336' (FIG. 7B). The
chipannel 710 may be made of optically clear resin such as COC, COP
and PMMA.
[0174] In some embodiments, the nucleic acid-based detection probes
are printed on the detection area 333' of the chipannel 710 by UV
irradiation. In some examples, the detection area 333' further
comprises control probes immobilized thereon. The detection probes
and control probes are immobilized to form separate reaction panels
and control panels. In some embodiments, the nucleic acid probes
and control probes are printed on the detection area 333' of the
chipannel 710 as shown in FIG. 13C. The detection probes and
control probes are printed to the reaction panel 1312 and the
control panel 1313, respectively. Within each panel, the detection
probes and control probes are printed in a checkerboard pattern,
such as the pattern shown in FIG. 13D.
[0175] FIGS. 7C and 7D illustrate perspective views of the
chipannel 710. In one embodiment, the chipannel 710 is held by a
port connection 711 (FIG. 7C). A vacuum, for example, the vacuum of
the detection device 100 is connected to the chipannel 710 through
the port connection 711. In another embodiment, the chipannel 710
is sealed to the cup bottom 337 via a face seal 712 (FIG. 7D). The
overmolding of the chipannel 710 and the cup bottom 330 will result
in a seamless combination of the parts. Any overmolding and casting
techniques, e.g., an injection molding process, may be used to
overmold the parts into a single part.
[0176] In some embodiments, the solid substrate with detection
probes immobilized thereon (e.g., chipannel 710) may be a glass
with a high optical clarity such as borosilicate glass and soda
glass.
[0177] In other embodiments, the solid substrate with detection
probes immobilized thereon (e.g., chipannel 710) may be made of
plastic materials high optical clarity. As non-limiting example,
the substrate may be selected from the group consisting of
polydimethylsiloxane (PDMS), cyclo-olefin copolymer (COC),
polymethylmetharcylate (PMMA), polycarbonate (PC), cyclo-olefin
polymer (COP), polyamide (PA), polyethylene (PE), polypropylene
(PP), polyphenylene ether (PPE), polystyrene (PS), polyoxymethylene
(POM), polyetheretherketone (PEEK), polytetrafluoroethylene (PTFE),
polyvinylchloride (PVC), polyvinylidene fluoride (PVDF),
polyvinylalcohol, polyacylate, polybutyleneterephthalate (PBT),
fluorinated ethylenepropylene (FEP), perfluoralkoxyalkane (PFA),
polypropylene carbonate (PPC), polyether sulfone (PES),
polyethylene terephthalate (PET), cellulose, poly(4-vinylbenzyl
chloride) (PVBC), Toyopearl.RTM., hydrogels, polyimide (PI),
1,2-polybutadiene (PB), fluoropolymers- and copolymers (e.g.
poly(tetrafluoroethylene) (PTFE), perfluoroethylene propylene
copolymer (FEP), Ethylene tetrafluoroethylene (ETFE)), polymers
containing norbomene moieties, polymethylmethacrylate, acrylic
polymers or copolymers, polystyrene, substituted polystyrene,
polyimide, silicone elastomers, fluoropolymers, polyolefins,
epoxies, polyurethanes, polyesters, polyethylene terephtalate,
polypersulfone, and polyether ketones, and a combination thereof.
The chips and chipannel may be prepared with injection mold. In
another embodiment of the test cup 300 shown FIG. 7E, the cup is
further optimized to improve its performance and for manufacture.
In this embodiment, the filter gasket 623 is overmolded to the
interior of the cup body, e.g., in the homogenization chamber 321
(FIG. 7F). FIG. 7H demonstrates a cross-sectional view of the
overmolded seal 713 that combines the parts into one single part.
The overmolding facilitates the manufacturing process to result in
a single piece. In this embodiment, the top of the valve shaft 351
of the rotary valve 350 comprises a cam 353 (FIG. 7G) that
interacts with the filter cap 621 to provide a rotating motion
(FIG. 7F, right panel). FIG. 7I demonstrates the cup bottom 337
(the top panel) and the bottom perspective view of the cup body 320
(the bottom panel). In this embodiment, the rotary valve 350 is
secured in the test cup body 320 through a plurality of compression
coil springs 721 located at the cup bottom cover 337 (FIG. 7J).
FIG. 7J further demonstrates the compression coil springs 721 at
the cup bottom 337. Four coil springs 721 may locate at the corners
of the rotary valve port 350a to secure the valve 350. In this
embodiment, the chipannel 710 may be welded to the bottom of the
cup body 320. For example, the chipannel 710 may be laser welded to
the bottom of the cup body 320. FIG. 7K demonstrates, in one
example, the weld bead materials 722 at the bottom of the cup body
320 for laser welding.
[0178] The cup bottom 330 is configured to close the disposable
test cup 300 and to provide a means for coupling the test cup 300
to the detection device 100 in various embodiments discussed
herein. In some embodiments, the bottom side of the bottom assembly
330 of the cup 300 shown in FIG. 3H, includes several interfaces
for connecting the cup 300 to the detection device 100 for
operation, including a homogenization rotor interface 340a that may
couple the homogenization rotor 340 to a motor in the device 100
for controlling homogenization; the valve interface 350a that may
couple the rotary valve 350 to a motor in the device 100 for
controlling valve rotation; and a pump interface 380 for connecting
to a pump in the detection device 100.
[0179] In some embodiments, a valve system is provided to control
the fluid flow of the sample, detection agents, buffers and other
reagents, and waste through different parts of the cartridge (e.g.,
separate chambers within the cup). In addition to flexible
membranes, foil seals and pinch valves discussed herein, other
valves may be included to control the flow of the fluid during the
process of a detection assay, including swing check valves, gate
valves, ball valves, globe valves, rotary valves, custom valves, or
other commercially available valves. For example, a gland seal or
rotary valve 350 may be used to control the flow of the processed
sample solution within the cup 300. In some examples, pinch valves
or rotary valves are used to completely isolate the fluid from
other internal valve parts. In other examples, air operated valves
(e.g., air operated pinch valves) may be used to control the fluid
flow, which are operated by a pressurized air supply.
[0180] In one embodiment, means for controlling the fluid flow
within the cup chambers may be included in, for example, the cup
bottom assembly 330 and/or the cup body 320. The means may comprise
flow channels, tunnels, valves, gaskets, vents and air connections.
In other embodiments, the means for the fluid flow may be
configured as a separate component in the cup, e.g., the fluidic
panel 631 shown in FIG. 6A.
[0181] In other embodiments, the valve system of the present
disclosure may comprise additional air vents included in the test
cup 300, to control air flow when the DNA coated glass chip is used
as the detection sensor. The DNA chip may be purged by air during
the procession of an allergen detection assay. Individual air
intakes may be opened based on the requirement of the system. The
valve system as discussed herein may be used to keep the air vent
unit inactive until use. The air port(s) allow air into the
cartridge (e.g., the cup 300) and the air vent(s) allow air to
enter various chambers when fluids are added to the chambers or
removed from the chambers. The air vents may also have a membrane
incorporated in them to prevent spillage and to act as a mechanism
to control fluid fill volumes by occlusion of the vent membrane
thus stopping further flow and fill function.
[0182] In one preferred embodiment, the rotary valve 350 (shown in
FIG. 3C, FIG. 5B, FIGS. 6A and 6F and FIG. 7A) may be used to
control and regulate fluid flow and rate in the test cup 300. The
rotary valve 350 comprising a valve shaft 351 and a valve disc 352
(FIG. 6F and FIG. 7G) can be operated by an associated detection
device (e.g., the device 100). In some embodiments, the rotary
valve 350 may position at a specific angle by rotating the valve
components either counterclockwise (CCW) or clockwise (CW) at each
step of the repeated washing and air purge cycle(s) during the
process of a detection assay. The air hole can allow air in. Air is
drawn through the system via vacuum pressure to perform air purge
functions. The angle may range from about 2.degree. to about
75.degree..
[0183] As a non-limiting example, the valve may be at about
38.5.degree. as reference from the air hole wherein the pump 1040
is off and the reaction chamber 331 is dry (referred to as home
position). After the test sample is processed and homogenized, the
pump is on and the valve 350 is rotated CCW and parks at an angle
of about 68.5.degree., allowing the processed sample to be
transported to the filtration chamber 322. Next, the valve
components may be rotated again at different directions to park at
different angles such as about 57.degree. to flow wash buffer to
the reaction chamber 331, and about 72.degree. to purge the DNA
chip with air. After the prewash of the DNA chip, the valve
components may be rotated to the home position at about
38.5.degree.. The processed sample solution is pulled through the
filter assembly 325. After filtration, the valve components may be
rotated and park at an angle of about 2.degree., allowing the
collected filtrate to flow into the reaction chamber 331, wherein
the chemical reactions occur. The valve 350 will rotate and park at
about 57.degree. to flow wash buffer to the reaction chamber 331,
and park at about 72.degree. to purge the DNA chip with air. The
wash and air purge steps may be repeated one or more times until
the optical measuring indicates a clean background.
[0184] In other embodiments, the rotary valve 350 is operatively
connected to a filter cap 621 (FIG. 6E). the filter cap locks the
rotary valve 350, for example during the shipment of the test cup
300.
[0185] In one embodiment, the valve system may be a rotary valve as
shown in FIG. 8A and FIG. 8B. In this embodiment, the rotary valve
350 is positioned to control air in and fluid flow. The positioning
may drive the homogenization in the homogenization chamber 321,
filtration and collection of filtrate (F), sample washes (e.g.,
wash 1-(W1) and wash 2 (W2) and waste collection (in FIG. 8A). In
step 1 of FIG. 8B, the rotary valve 350 is in a closed position
with no connections being made between any of the chambers. In step
2 of FIG. 8B, the rotary valve 350 connects the wash 1 chamber W1
to the reaction chamber 331 to flush the reaction chamber 331 with
the wash buffer subsequently being pushed out to the waste chamber
323. In step 3 of FIG. 8B, the rotary valve 350 connects the
homogenization chamber 321 to the filtrate chamber F to affect the
filtration step. In step 4 of FIG. 8B, the rotary valve 350
connects the filtrate chamber F to the reaction chamber 331 to send
the filtrate to the reaction chamber 331 for reaction and analysis.
In step 5 of FIG. 8B, the rotary valve 350 connects the wash 2
chamber W2 to the reaction chamber to flush the reaction chamber
331 again.
[0186] In some embodiments, extraction buffers may be pre-stored in
the analytic cartridge, e.g., the homogenization chamber 321 of the
cup body 320, for example in foil sealed reservoirs like the food
processing reservoir 801 (FIG. 8C). Alternatively, extraction
buffers may be stored separately in a separate buffer reservoir in
the cup body 320, a reservoir similar to the wash buffer storage
reservoir 802 (in the buffer storage chamber 324 (optional) as
shown in FIG. 8C). The extraction buffer after sample
homogenization and washing waste may be stored in the separate
waste reservoir 803 within the waste chamber 323. The waste chamber
323 has sufficient volume to store a volume greater than the amount
of fluid used during the detection assay.
[0187] In accordance with the present disclosure, the
homogenization rotor 340 may be constructed to be small enough to
fit into a disposable test cup 300, particularly into the
homogenization chamber 321, where the homogenizer processes a
sample to be tested. Additionally, the homogenization rotor 340 may
be optimized to increase the efficacy of sample homogenization and
protein extraction. In one embodiment, the homogenization rotor 340
may comprise one or more blades or the equivalent thereof at the
proximal end. In some examples, the rotor 340 may comprise one,
two, three or more blades. The homogenization rotor 340 is
configured to pull the test sample from the food corer 200 into the
bottom of the homogenization chamber 321.
[0188] Alternatively, the homogenization rotor 340 may further
comprise a center rod running through the rotor that connects
through the cup body 320 to a second interface bit. The central rod
may act as an additional bearing surface or be used to deliver
rotary motion to the rotor 340. When the rotor 340 is mounted to
the cup body through the port at the cup bottom (e.g., 340a), the
blade tips may remain submersed within the extraction buffer during
operation. In another alternative embodiment, the homogenization
rotor 340 may have an extension to provide a pass through the
bottom of the cup; the pass may be used as a second bearing support
and/or an additional location for power transmission. In this
embodiment, the lower part of the rotor has a taper to fit to a
shaft, forming a one-piece rotor. In accordance with the present
disclosure, depth of the blades of the homogenization rotor 340,
with or without the center rod, is constructed to ensure the blade
tips in the fluid during sample processing.
[0189] As compared to other homogenizers (e.g., U.S. Pat. No.
6,398,402; incorporated herein by reference in its entirety), the
custom blade core of the present disclosure spins and draws and
forces food into the toothed surfaces of the custom cap. The
homogenizer rotor may be made of any thermoplastics, including, but
not limited to, polyamide (PA), acrylanitrilebutadienestyrene
(ABS), polycarbonate (PC), high Impact polystyrene (HIPS), and
acetal (POM).
[0190] The disposable cartridge may be in any shape, for example,
circular, oval, rectangular, or egg-shaped. Any of these shapes may
be provided with a finger cut or notch. The disposable cartridge
may be asymmetrical, or symmetrical.
[0191] Optionally, a label or a foil seal may be included on the
top of the cup lid 311 to provide a final fluid seal and
identification of the test cup 300. For example, a designation of
peanut indicates that the disposable test cup 300 is used for
detecting the peanut allergen in a food sample.
The Detection Device
[0192] In some embodiments, the detection device 100 may be
configured to have an external housing 101 that provides support
surfaces for the components of the detection device 100; and a lid
103 that opens the detection device 100 for inserting a disposable
test cup 300 and covers the cup during operation. The small lid may
be located at one side of the device (as shown in FIG. 1 and FIG.
9A), or in the center (not shown). In some aspects of the
disclosure, the lid may be transparent, allowing all the operations
visible through the lid 103. The device may also comprise s USB
port 105 for transferring data.
[0193] One embodiment of the allergen detection device 100
according to the present disclosure is depicted in FIG. 1 and FIG.
9A. As illustrated in FIG. 1, the detection device 100 comprising
an external housing 101 that provides support for holding the
components of the detection device 100 together. The external
housing 101 may be formed of plastic or other suitable support
material. In other embodiments, the device may be made of Aluminum.
The device also has a port or receptacle 102 for docking the test
cup 300 (FIG. 1 and FIG. 9A).
[0194] To execute an allergen detection test, the detection device
100 is provided with a means (e.g., a motor) for operating the
homogenization assembly and necessary connectors that connect the
motor to the homogenization assembly; means (e.g., a motor) for
controlling the rotary valve; means for driving and controlling the
flow of the processed sample solution during the process of the
allergen detection test; an optical system; means for detecting
fluorescence signals from the detection reaction between the
allergen in the test sample and the detection agents; means for
visualizing the detection signals including converting and
digitizing the detected signals; a user interface that displays the
test results; and a power supply.
[0195] As viewed from the transparent lid 103 (FIG. 9A), the device
100 has an interface comprising areas for coupling the components
of the cartridge 300 (when inserted) for operating a detection
reaction (FIG. 9B). These areas include a homogenization bit 910
for coupling the rotor 340 to the motor, a vacuum bit 920 for
coupling the cup with the vacuum pump, a rotary valve drive bit 930
for coupling the rotary valve 350 to a valve motor and a protective
glass 940 which is aligned to the glass chip 333 or the sensor area
333' of the chipannel 710 through the optical window of the
reaction chamber 331. A data chip reader 950 is also included to
read the data chip 335. The pins 960 are used to facilitate
placement of the cup 300 in the receptacle of the device 100.
[0196] In one embodiment of the present disclosure, as shown in
FIG. 10A, the components of the detection device 100 that are
integrated to provide all motion and actuation for operating a
detection reaction, include a motor 1010 which may be connected to
the homogenization rotor 340 inside the homogenization chamber 321
within the cup body 320. The motor 1010 may be connected through a
multiple-component coupling assembly including a gear train/drive
platen for driving the rotor during homogenization in an allergen
detection test; a valve motor 1020 for driving the rotary valve
350; an optical system 1030 that is connected to the reaction
chamber 331 (not shown) or the chipannel 710 within the disposable
test cup 300; a vacuum pump 1040 for controlling and regulating air
and fluid flow (not shown in FIG. 10A), a PCB display 1050, and a
power supply 1060 (in FIG. 10B). A means for retaining the test cup
(i.e. the cup retention 1070) is included for holding the test cup
300. Each part is described below in detail.
1. Homogenization Assembly
[0197] In one embodiment, the motor 1010 may be connected to the
homogenization rotor 340 inside the test cup 300 through the
multiple-component rotor coupling assembly. The rotor coupling
assembly may include a coupling that is directly linked to the
distal end cap of the rotor 340, and a gearhead that is part of a
gear train or a drive (not shown) for connection to the motor 1010.
In some embodiments, the coupling may have different sizes at each
end, or the same sizes at each end of the coupling. The distal end
of the coupling assembly may connect to the rotor 340 through the
rotor port 340a at the cup bottom 330. It is also within the scope
of the present disclosure that other alternative means for
connecting the motor to the homogenization rotor 340 may be used to
form a functional homogenization assembly.
[0198] In some embodiments, the motor 1010 can be a commercially
available motor, for example, Maxon motor systems: Maxon RE-max
and/or Maxon A-max (Maxon Motor ag, San Mateo, Calif., USA).
[0199] Optionally, a heating system (e.g. resistance heating, or
peltier heaters) may be provided to increase the temperature of
homogenization, therefore, to increase the effectiveness of sample
dissociation and shorten the processing time. The temperature may
be increased to between 60.degree. C. to 95.degree. C., but below
95.degree. C. Increased temperature may also facilitate the binding
between detection molecules and the allergen being detected.
Optionally a fan or peltier cooler may be provided to bring the
temperature down quickly after implementing the test.
[0200] The motor 1010 drives the homogenization assembly to
homogenize the test sample in the extraction buffer and
dissociate/extract allergen proteins. The processed sample solution
may be pumped or pressed through the flow tubes to next chamber for
analysis, for example, to the reaction chamber 331 in which the
processed sample solution is mixed with the pre-loaded detection
molecules (e.g., aptamer-magnetic bead conjugates) for the
detection test. Alternatively, the processed sample solution may
first be pumped or pressed through the flow tubes to the filter
assembly 325 and then to the filtrate chamber 322 before
transported to the reaction chamber 331 for analysis.
2. Filtration
[0201] In some embodiments, means for controlling the filtration of
the processed test sample may be included in the detection device.
The food sample will be pressed through a filter membrane or a
filtering assembly before the extraction solution being delivered
to the reaction chamber 331, and/or other chambers for further
processing such as washing. One example is the filter membrane(s).
The membranes provide filtration of specific particles from the
processed protein solution. For example, the filter membrane may
filter particles up to from about 0.1 .mu.m to about 1000 .mu.m, or
about 1 .mu.m to about 600 .mu.m, or about 1 .mu.m to about 100
.mu.m, or about 1 .mu.m to about 20 .mu.m. In some examples, the
filter membrane may remove particles up to about 20 .mu.m, or about
19 .mu.m, or about 18 .mu.m, or about 17 .mu.m, or about 16 .mu.m,
or about 15 .mu.m, or about 14 .mu.m, or about 13 .mu.m, or about
12 .mu.m, or about 11 .mu.m, or about 10 .mu.m, or about 9 .mu.m,
or about 8 .mu.m, or about 7 .mu.m, or about 6 .mu.m, or about 5
.mu.m, or about 4 .mu.m, or about 3 .mu.m, or about 2 .mu.m, or
about 1 .mu.m, or about 0.5 .mu.m, or about 0.1 .mu.m. In one
example, the filter membrane may remove particles up to about 1
.mu.m from the processes sample. In some aspects, filter membranes
may be used in combination to filter specific particles from the
assay for analysis. This filter membrane may include multistage
filters. Filter membranes and/or filter assemblies may be in any
configuration relative to the flow valve. For example, the flow
valves may be above, below or in between any of the stages of the
filtration.
[0202] In some embodiments, the filter assembly may be a complex
filter assembly 325 as illustrated in FIG. 4A in which the
processed sample is filtered sequentially through the gross filter
411, the depth filter 412 and the membrane filter 420. In other
embodiments, the filter assembly 325 may the filter stack shown in
FIG. 6D.
3. Pump and Fluid Motion
[0203] In accordance with the present disclosure, a means for
driving and controlling the flow of the processed sample solution
is provided. In some embodiments, the means may be a vacuum system
or an external pressure. As a non-limiting example, the means may
be a platen (e.g., a welded plastic clamshell) configured to being
multifunctional in that it could support the axis of the gear train
and it could provide the pumping (sealed air channel) for the
vacuum to be applied from the pump 1040 to the test cup 300. The
pump 1040 may be connected to the test cup 300 through the pump
port 920 located at the bottom (FIG. 9B), which connects to the
pump interface 380 (FIG. 3G) on the bottom 330 of the test cup 300
when the cup is inserted to the device.
[0204] The pump 1040, such as piezoelectric micro pump (e.g.,
Takasago Electric, Inc., Nagoya, Japan), or peristaltic pump, may
be used to control and automatically adjust the flow to a target
flow rate. The flow rate of a pump is adjustable by changing either
the driver voltage or drive frequency. As a non-limiting example,
the pump 1040 may be a peristaltic pump. In another embodiment, the
pump 1040 may be is a piezo pump currently on the market that have
specifications that indicate they could be suitable for the aliquot
function required to flow filtered sample solution to different
chambers inside the test cup 300. The pump 1040 may be a vacuum
pump or another small pump constructed for laboratory use such as a
KBF pump (KNF Neuberger, Trenton, N.J., USA).
[0205] Alternatively, a syringe pump, diaphragm and/or a
micro-peristaltic pump may be used to control fluid motion during
the process of a detection assay and/or supporting fluidics. In one
example, an air operated diaphragm pump may be used.
4. Rotary Valve Control
[0206] In some embodiments, the rotary valve 350 (e.g., as shown in
FIG. 6F) for controlling fluid flow needs to be in precise
positions. A means to control the rotary valve is provided and the
control mechanism is able to rotate the valve in both directions
and accurately stop at desired locations. In some embodiments, the
device 100 includes a valve motor 1020 (in FIG. 10A). As shown in
FIG. 11A, the valve motor 1020 may be a low cost, DC geared motor
1110 with two low cost optical sensors (1131 and 1132), and a
microcontroller. An output coupling 1120 interfaces with the rotary
valve 350. In some embodiments, the output coupling 1120 has a
`half-moon` shelf 1170 as shown in FIG. 11B, which interrupts the
output optical sensor 1131 with the protruding half. The output
optical sensor signal toggles between high and low, depending on
whether or not the protruding shelf interrupts the sensor. A
microcontroller (MCU) detects these transitions and get an absolute
position of the output from this signal. The positioning of these
transitions is important and application specific since these
transitions are used during directional changes to account for gear
backlash.
[0207] The direct motor shaft 1140 has a paddle wheel which
interrupts the direct shaft optical sensor 1132, allowing the
direct shaft optical sensor 1132 to output a train of pulses, with
the number of pulses per revolution determined by the number of
paddles on the wheel 1150. The MCU reads this train of pulses and
determines the degrees movement of the output coupling. The
resolution is dependent on the number of paddles of the direct
shaft encoder wheel 1150, and the gear reduction ratio of the gear
box 1160.
[0208] The MCU interprets the output of these two optical sensors
and can drive the output to a desired location, as long as the
position of the output coupling shelf transitions, the number of
paddle wheels on the direct encoder wheel 1120, and the gear ratio
are known. During a change of direction, the motor must rotate by a
fixed amount before an output transition is seen, the fixed amount
is selected to overcome backlash of the gears. Once the fixed
amount is overcome, on the next output signal transition, the MCU
can start counting the direct signal pulses with confidence that
they correspond to accurate output of location and movement.
5. Optical System
[0209] The detection device 100 of the present disclosure comprises
an optical system that detects optical signals (e.g., a
fluorescence signal) generated from the interaction between an
allergen in the sample and detection agents (e.g., aptamers and
SPNs). The optical system may comprise different components and
variable configurations depending on the types of the fluorescence
signal to be detected. The optical system is close to and aligned
with the detection cartridge, for instance, the primary optical
window and optionally the secondary optical window of the reaction
chamber 331 of the test cup 300 as discussed above.
[0210] In some embodiments, the optical system 1030 may include
excitation optics 1210 and emission optics 1220 (FIGS. 12A and
12B). In one embodiment, as shown in FIG. 12A, the excitation
optics 1210 may comprise a Light Emitted Diode (LED) 1211
configured to transmit an excitation optical signal to the sensing
area (e.g., 332) in the reaction chamber 331, a collimation lens
1212 configured to focus the light from the light source, a filter
1213 (e.g., a bandpass filter), a focus lens 1214, and an optional
LED power monitoring photodiode. The emission optics 1220 may
comprise a focus lens 1221 configured to focus at least one portion
of the allergen-dependent optical signal onto the detector
(photodiode), two filters including a longpass filter 1222 and a
bandpass filter 1223, a collection lens 1224 configured to collect
light emitted from the reaction chamber and an aperture 1225. The
emission optics collects light emitted from the solid surface (e.g.
a DNA chip 333) in the detection chamber 331 and the signal is
detected by the detector 1230 configured to detect an
allergen-dependent optical signal emitted from the sensing area
332. In some aspects, the excitation power monitoring may be
integrated into the LED (not shown in FIG. 12A).
[0211] A light source 1211 is arranged to transmit excitation light
within the excitation wavelength range. Suitable light sources
include, without limitation, lasers, semi-conductor lasers, light
emitting diodes (LEDs), and organic LEDs.
[0212] An optical lens 1212 may be used along with the light source
1211 to provide excitation source light to the fluorophore. An
optical lens 1214 may be used to limit the range of excitation
light wavelengths. In some aspects, the filter may be a band-pass
filter.
[0213] Fluorophore labeled SPNs specific to a target allergen are
capable of emitting, in response to excitation light in at least
one excitation wavelength range, an allergen-binding dependent
optical signal (e.g. fluorescence) in at least one emission
wavelength range.
[0214] In some embodiments, the emission optics 1220 are operable
to collect emissions upon the interaction between detection agents
and target allergens in the test sample from the reaction chamber
331. Optionally, a mirror may be inserted between the emission
optics 1220 and the detector 1230. The mirror can rotate in a wide
range of angles (e.g., from 1.degree. to 90.degree.) which could
facilitate formation of a compacted optical unit inside the small
portable detection device.
[0215] In some embodiments, more than one emission optical system
1220 may be included in the detection device. As a non-limiting
example, three photodiode optical systems may be provided to
measure fluorescence signals from an unknown test area and two
control areas on a glass chip (e.g., see FIG. 13B). In other
aspects, an additional collection lens 1224 may be further included
in the emission optics 1220. This collection lens may be configured
to detect several different signals from the chip 333. For example,
when the detection assay is implemented using a DNA glass chip,
more than two control areas may be constructed on the solid surface
in addition to a detection area for allergen detection. The
internal control signals from each control area may be detected at
the same time when an allergen derived signal is measured. In this
context, more than two collection lenses 1224 may be included in
the optical system 1030, one lens 1224 for signal from the
detection area and the remaining collection lenses 1224 for signals
from the control areas.
[0216] The detector (e.g., photodiode) 1230 is arranged to detect
light emitted from the fluidic chip in the emission wavelength
range. Suitable detectors include, without limitation, photodiodes,
complementary metal-oxide-semiconductor (CMOS) detectors,
photomultiplier tubes (PMT), microchannel plate detectors, quantum
dot photoconductors, phototransistors, photoresistors, active-pixel
sensors (APSs), gaseous ionization detectors, or charge-coupled
device (CCD) detectors. In some aspects, a single and/or universal
detector can be used.
[0217] In some embodiments, the detector 1230 may be an image
detector, such as a camera as described hereinbelow.
[0218] In some embodiments, the optical system 1030 may be
configured to detect fluorescence signals from the solid substrate
sensor (e.g., DNA chip 333 shown in FIG. 13A or the chipannel 710
shown in FIGS. 7A to 7C). The DNA chip may be configured to contain
a central reaction panel which is marked as an "unknown" signal
area on the chip (FIG. 13A), and at least two control areas at
various locations of the chip (FIG. 13A). In this context, the
optical system 1030 is configured to measure both detection signals
and internal control signals simultaneously (FIG. 13B).
[0219] In one example, the optical system 1030 comprises two
collection lenses 1224 and corresponding optical components, such
as control array photodiodes for each lens 1224. FIG. 12B
demonstrates a side view of the optical system 1030 shown in FIG.
12A inside the detection device 100. In this embodiment, two
collection lenses 1224 are included in the optical system, one for
collecting control array signals from the DNA chip (e.g., the two
signals 1301 and 1302 shown in FIG. 13B) and one specific to the
unknown detection signal from the DNA chip (e.g., the detection
signal 1302 as shown in FIG. 13B). In other aspects, the collection
lenses 1224 may be configured to collecting signals from the
detection area 333' of the chipannel 710, e.g., one signal from the
reaction panel 1312 and the other signal from the control panel
1313 shown in FIG. 13C. A signal array diode 1241 (e.g., the LED
diode 1211 shown in FIG. 12A) and two control assay photodiodes
1242 are included for each optical path. Additionally, two prisms
1243 may be added to the two collection-lenses (1224) configured
for collecting signals from the two control areas. The prisms 1243
can bend the control array light to the photodiode sensor area.
[0220] In some embodiments, the optical system 1030 may be
configured as a straight mode as shown in FIG. 14A. The excitation
optics 1410, which are configured to transmit an excitation optical
signal to the glass chip 333 (e.g., DNA coated chip) in the
reaction chamber 331, may comprise a LED 1411, a collimation lens
1412, a bandpass filter 1413 and a cylinder lens 1414. The cylinder
lens 1414 may cause the excitation light to form a line to cover
the reaction panel and control panels on the glass chip (e.g., FIG.
13B). The emission optics 1420 which are aligned with the glass
chip 333 may comprise a collection lens 1421 configured to collect
light emitted from the glass chip 333, a bandpass filter 1422a, a
longpass filter 1422b, and a focus lens 1423 configured to focus at
least one portion of the allergen-dependent optical signal onto the
chip reader 1430. The chip reader 1430 is composed of three
photodiode lenses 1431, two control array photodiodes 1432, a
signal array photodiode 1433 and a collection PCB 1434 (FIG. 14A).
In some embodiments, the collection lens 1421 may be shaped to
contain a concave first surface to optimize imaging and minimize
stray light.
[0221] As a non-limiting example, the excitation optics 1410 and
the emission optics 1420 may be folded and configured into a
stepped bore 1480 in the device 100 (see FIG. 14C). An excitation
folding mirror 1440 and a collection folding mirror 1450 may be
configured to minimize the light paths from the excitation optics
1410 and the emission optics 1420, respectively (in FIG. MB). The
minimized volume can modulate the laser at a frequency to minimize
interference from environmental light sources. A photodiode shield
1460 may be added to cover and protect the chip reader 1430 shown
in FIG. 14A. The reader 1430 is then positioned close to the
collection lens 1421 to minimize the scattered light. FIG. 14C
illustrates an example of the stepped bore 1480 in the device to
hold the emission optics 1420. The aperture 1470 of the collection
lens 1421 is shown in FIG. 14C.
[0222] The LED source (e.g., 1411) may be modulated, and/or
polarized and oriented to minimize the reflections from the glass
chip. Accordingly, the chip reader may be synchronized to measure
modulated light.
[0223] FIG. 15A illustrates another embodiment of the optical
system 1030. In this embodiment, the optical system 1030 comprises
an image detector. The image detector may be a camera 1531, as part
of the signal reader 1530. The camera may catch the reaction images
of the sensor DNA chip 333 or the detection area 333' of the
chipannel 710. As a non-limiting example, the optical system 1030
shown in FIG. 15A, comprises an excitation optics 1510 comprising
excitation filter 1513, collimation lens 1512 and laser diode 1511,
an emission optics 1520 comprising a collection lens 1521, bandpass
filter 1522a, longpass filter 1522b (e.g., color glass longpass
filter) and focus lens 1523, and a signal reader 1530 comprising a
camera 1531. Each system of the optical system may be configured in
an optical housing, e.g., the optical housing 1540 in FIG. 15A
configured for holding the components of the emission optics
1520.
[0224] FIG. 15B illustrates a cross-sectional view of the optical
system of FIG. 15A assembled inside the detection device 100. From
this cut-away side view, the excitation optics 1510 and the
emission optics 1520 are assembled into an optical housing,
respectively. A protective window 1501 may be added to protect the
optical components. Optionally, a laser adjustment mount 1502 may
be included to adjust the laser diode 1511 inside the excitation
optics 1510. The camera 1531 catches the reaction images and the
raw images are collected and processed. The detection results may
be displayed through the display PCB 1050.
[0225] The above described optical system 1030 is illustrative
examples of certain embodiments. Alternative embodiments might have
different configurations and/or different components.
[0226] In other embodiments, a computer or other digital control
system can be used to communicate with the light filters, the
fluorescence detector, the absorption detector and the scattered
detector. The computer or other digital control systems control the
light filter to subsequently illuminate the sample with each of the
plurality of wavelengths while measuring absorption and
fluorescence of the sample based on signals received from the
fluorescence and absorption detectors.
6. Display
[0227] As shown in a cut-away side view in FIG. 10B, a printed
circuit board (PCB) 1050 is connected to the optical system 1030.
The PCB 1050 may be configured to be compact with the size of the
detection device 100 and at the same time, may provide enough space
to display the test result.
[0228] Accordingly, the test result may be displayed with back lit
icons, LEDs or an LCD screen, OLED, segmented display or on an
attached cell phone application. The user may see an indicator that
the sample is being processed, that the sample was processed
completely (total protein indictor) and the results of the test.
The user may also be able to view the status of the battery and
what kind of cartridge is placed in the device (bar code on the
cartridge or LED assembly). The results of the test will be
displayed, for example, as (1) actual number ppm or mg; or (2)
binary result yes/no; or (3) risk analysis--high/medium/low or
high/low, risk of presence; or (4) range of ppm less than 1/1-10
ppm/more than 10 ppm; or (5) range of mg less than 1 mg/between
1-10 mg/more than 10 mg. The result might also be displayed as
number, colors, icons and/or letters.
[0229] In accordance with the present disclosure, the detection
device 100 may also include other features such as means for
providing a power supply and means for providing control of the
process. In some embodiments, one or more switches are provided to
connect the motor, the micropump and/or the gear train or the drive
to the power supply. The switches may be simple microswitches that
can turn the detection device on and off by connecting and
disconnecting the battery.
[0230] The power supply 1060 may be a Li-ion AA format battery or
any commercially available batteries that are suitable for
supporting small medical devices such as the Rhino 610 battery, the
Turntigy Nanotech High dischargeable Li Po battery, or the Pentax
D-L163 battery.
[0231] In the description herein, it is understood that all recited
connections between components can be direct operative connections
or indirectly operative connections. Other components may also
include those disclosed in the applicant's U.S. Provisional
application 62/461,332, filed on Feb. 21, 2017; the contents of
which are incorporated herein by reference in their entirety.
Detection Assays
[0232] In another aspect of the present disclosure, provided is an
allergen detection test implemented using detection assemblies and
systems, detection agents and detection sensors of the present
disclosure.
[0233] As a non-limiting example, an allergen detection test
comprises the steps of (a) collecting a certain amount of a test
sample suspected of containing an allergen of interest, (b)
homogenizing the sample and extracting allergen proteins using an
extraction/homogenization buffer, (c) contacting the processed
sample with a detection agent that specifically binds to a target
allergen; (d) contacting the mixture in (c) with a detection sensor
comprising a solid substrate that is printed with nucleic acid
probes; (e) measuring fluorescence signals from the reaction; and
(f) processing and digitizing the detected signals and visualizing
the interaction between the detection agents and the allergen.
[0234] In some aspects of the disclosure, the method further
comprises the step of washing off the unbound compounds from the
detection sensor to remove any non-specific binding
interactions.
[0235] In some aspects of the disclosure, the method further
comprises the step of filtering of the processed sample prior to
contacting it with the detection sensor (e.g., DNA chip).
[0236] In some embodiments, an appropriately sized test sample is
collected for the detection assay to provide a reliable and
sensitive result from the assay. In some examples, a sampling
mechanism that can collect a test sample effectively and
non-destructively for fast and efficient extraction of allergen
proteins for detection is used.
[0237] A sized portion of the test sample can be collected using,
for example, a food corer 200 illustrated in FIG. 2B. The food
corer 200 collect an appropriately sized sample from which can be
extracted sufficient protein for the detection test. The sized
portion may range in mass from 0.1 g to 1 g, preferably 0.5 g.
Furthermore, the food corer 200 may pre-process the collected test
sample by cutting, grinding, blending, abrading and/or filtering.
Pre-processed test sample will be introduced into the
homogenization chamber 321 for processing and allergen protein
extraction.
[0238] The collected test sample is processed in an
extraction/homogenization buffer. In some aspects, the extraction
buffer is stored in the homogenization chamber 321 and may be mixed
with the test sample by the homogenization rotor 340. In other
aspects, the extraction buffer may be released into the
homogenization chamber 321 from another separate storage chamber.
The test sample and the extraction buffer will be mixed together by
the homogenization rotor 340 and the sample being homogenized. In
some embodiments, the extraction buffer is preloaded with a
detection agent (e.g., SPN), thereby permitting the extracted
molecule of interest from the test sample to interact with the
detection agent.
[0239] The extraction buffer may be universal target extraction
buffer that can retrieve enough target proteins from any test
sample and be optimized for maximizing protein extraction. In some
embodiments, the formulation of the universal protein extraction
buffer can extract the protein at room temperature and in minimal
time (less than 1 min). The same buffer may be used during food
sampling, homogenization and filtering. The extraction buffer may
be PBS based buffer containing 10%, 20% or 40% ethanol, or Tris
based buffer containing Tris base pH8.0, 5 mM MEDTA and 20%
ethanol, or a modified PBS or Tris buffer. In some examples, the
buffer may be a HEPES based buffer. Some examples of modified PBS
buffers may include: P+ buffer and K buffer. Some examples of Tris
based buffers may include Buffer A+, Buffer A, B, C, D, E, and
Buffer T. As a non-limiting example, the extraction buffer may
include 20 mM EPPS, 2% PEG 8000, 2% F-127 (Pluronic), 0.2% Brij-58
(pH8.4). In some embodiments, the extraction buffer may be
optimized for increasing protein extraction. A detailed description
of each modified buffer is disclosed in the PCT Patent Application
No.: PCT/US2014/062656; the content of which is incorporated herein
by reference in its entirety.
[0240] In accordance with the present disclosure, MgCl.sub.2 is
added after the sample is homogenized. In some embodiments,
MgCl.sub.2 solution (e.g., 30 .mu.L of 1M MgCl.sub.2 solution) is
added to the homogenization chamber (e.g., 321 in FIG. 3F) after
the sample homogenization.
[0241] In other embodiments, solid MgCl.sub.2 formulations may be
used in replacement of the addition of MgCl.sub.2 solution during
the reaction. The solid formulation may be provided as a MgCl.sub.2
lyophilized pellet in the homogenization chamber (e.g., 321 in FIG.
3F) which is dissolved by the homogenate after filtration, or a
filter component deposited or layered in the filter (e.g., the
filter membrane 420 in FIG. 4A and the filter assembly 325 in FIG.
4A and FIG. 6D) that is dissolved by the homogenate during the
filtration, or a MgCl.sub.2 film deposited on the inner surface of
the homogenization chamber 321), or MgCl.sub.2 containing
lyophilized beads stored in the filtrate chamber (e.g., the
filtrate chamber 322) or on a separate support. In the context of
the filter assembly 325, the cotton layer filter of the depth
filter (e.g., 412) may be impregnated with the MgCl.sub.2
formulation. Regardless of the formulations, MgCl.sub.2 will
dissolve in less than 1 minute, preferably in less than 30 seconds,
to be contacted with the processed sample homogenate. MgCl.sub.2
may dissolve in about 10 seconds, or about 15 seconds, or about 20
seconds, or about 25 seconds, or about 30 seconds. The solid
formulation will release MgCl.sub.2 within this short period of
time to reach to a final concentration of 30 mM. In some aspects,
the solid MgCl.sub.2 formulation may not break up into powder.
[0242] The volume of the extraction buffer may be from 0.5 mL to
3.0 mL. In some embodiments, the volume of the extraction buffer
may be 0.5 mL, 1.0 mL, 1.5 mL, 2.0 mL, 2.5 mL or 3.0 mL. The volume
has been determined to be efficient and repeatable over time and in
different food matrices.
[0243] In accordance with the present disclosure, the test sample
is homogenized and processed using the homogenization assembly that
has been optimized with high speed homogenization for maximally
processing the test sample.
[0244] In some aspects of the disclosure, a filtering mechanism may
be linked to the homogenizer. The homogenized sample solution is
then driven to flow through a filter in a process to further
extract allergen proteins and remove particles that may interfere
with the flow and optical measurements during the test, lowering
the amount of other molecules extracted from the test sample. The
filtration step may further achieve uniform viscosity of the sample
to control fluidics during the assay. In the context that DNA glass
chips are used as detection sensors, the filtration may remove fats
and emulsifiers that may adhere to the chip and interfere with the
optical measurements during the test. In some embodiments, a filter
membrane such as cell strainer from CORNING (CORNING, N.Y., USA) or
similar custom embodiment may be connected to the homogenizer. The
filtering process may be a multi-stage arrangement with different
pore sizes from first filter to second, or to the third. The
filtering process may be adjusted and optimized depending on food
matrices being tested. As a non-limiting example, a filter assembly
with a small pore size may be used to capture particles and to
absorb large volumes of liquid when processing dry foods,
therefore, longer times and higher pressures may be used during the
filtration. In another example, bulk filtration may be implemented
to absorb fat and emulsifiers when processing fatty foods. The
filtration may further facilitate to remove fluorescence haze or
particles from fluorescence foods, which will interfere with the
optical measurements.
[0245] The filter may be a simple membrane filter, or an assembly
composed of a combination of filter materials such as PET, cotton
and sand, etc. In some embodiments, the homogenized sample may be
filtered through a filter membrane, or a filter assembly, e.g., the
filter assembly 325 in FIG. 4A.
[0246] In some aspects of the present disclosure, the sampling
procedure may reach effective protein extraction in less than 1
minute. In one aspect, speed of digestion may be less than 2
minutes including food pickup, digestion and readout.
Approximately, the procedure may last 15 seconds, 30 seconds, 45
seconds, 50 seconds, 55 seconds, 1 minute or 2 minutes.
[0247] Extracted allergen proteins may be mixed with one or more
detection agents that are specific to one or more allergens of
interest. The interaction between allergen protein extraction and
detection agents will generate a detectable signal which indicates
the presence, or absence or the amount of one or more allergens in
the test sample. As used herein, the term "detection agent" or
"allergen detection agent" refers to any molecule which is capable
of, or does, interact with and/or bind to one or more allergens in
a way that allows detection of such allergen in a sample. The
detection agent may be a protein-based agent such as antibody, a
nucleic acid-based agent or a small molecule.
[0248] In some embodiments, the detection agent is a nucleic acid
molecule based signaling polynucleotide (SPN). The SPN comprises a
core nucleic acid sequence that binds to a target allergen protein
with high specificity and affinity. The SPN may be derived from an
aptamer selected by a SELEX method. As used herein, the term
"aptamer" refers to a nucleic acid species that has been engineered
through repeated rounds of in vitro selection or equivalently,
SELEX (systematic evolution of ligands by exponential enrichment)
to bind to various molecular targets such as small molecules,
proteins, nucleic acids, and even cells, tissues and organisms. The
binding specificity and high affinity to target molecules, the
sensitivity and reproductively at ambient temperature, the
relatively low production cost, and the possibility to develop an
aptamer core sequence that can recognize any protein, ensure an
effective but simple detection assay.
[0249] In accordance with the present disclosure, SPNs that can be
used as detection agents may be aptamers specific to a common
allergen such as peanut, tree-nut, fish, gluten, milk and egg. For
example, the detection agent may be the aptamers or SPNs described
in applicants' relevant PCT application publication Nos.
WO2015066027, WO2016176203, WO2017160616 and WO2018089391; and U.S.
Provisional Application No: 62/714,102 filed Aug. 3, 2018; the
contents of each of which are incorporated herein by reference in
their entirety.
[0250] In some embodiments, the detection agent (e.g., SPN) may be
labeled with a fluorescence marker. The fluorescence marker,
fluorophore may suitably have an excitation maximum in the range of
200 to 700 nm, while the emission maximum may be in the range of
300 to 800 nm. The fluorophore may further have a fluorescence
relaxation time in the range of 1-7 nanoseconds, preferably 3-5
nanoseconds. As non-limiting examples, a fluorophore that can be
probed at one terminus of a SPN may include derivatives of
boron-dipyrromethene (BODIPY, e.g., BODIPY TMR dye; BODIPY FL dye),
fluorescein including derivatives thereof, rhodamine including
derivatives thereof, dansyls including derivatives thereof (e.g.
dansyl cadaverine), texas red, eosin, cyanine dyes,
indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine,
squaraines and derivatives seta, setau, and square dyes,
naphthalene and derivatives thereof, coumarin and derivatives
thereof, pyridyloxazole, nitrobenzoxadiazole, benzoxadiazole,
anthraquinones, pyrene and derivatives thereof, oxazine and
derivatives, nile red, nile blue, cresyl violet, oxazine 170,
proflavin, acridine orange, acridine yellow, auramine, crystal
violet, malachite green, porphin, phthalocyanine, bilirubin,
tetramethylrhodamine, hydroxycoumarin, aminocoumarin;
methoxycoumarin, cascade blue, pacific blue, pacific orange, NBD,
r-phycoerythrin (PE), red 613; perCP, trured; fluorX, Cy2, Cy3, Cy5
and Cy7, TRITC, X-rhodamine, lissamine rhodamine B, allophycocyanin
(APC) and Alexa fluor dyes (e.g., Alexa Fluo 488, Alexa Fluo 500,
Alexa Fluo 514, Alexa Fluo 532, Alexa Fluo 546, Alexa Fluo 555,
Alexa Fluo 568, Alexa Fluo 594, Alexa Fluo 610, Alexa Fluo 633,
Alexa Fluo 637, Alexa Fluo 647, Alexa Fluo 660, Alexa Fluo 680, and
Alexa Fluo 700).
[0251] In one example, the SPN is labeled with Cy5 at the 5' end of
the SPN sequence. In another example, the SPN is labeled with Alexa
Fluo 647 at the one end of the SPN sequence.
[0252] In some embodiments, the SPN specific to an allergen of
interest may be pre-stored in the extraction/homogenization buffer
in the homogenization chamber 321 (FIGS. 3B and 3F). The extracted
allergen protein, if present in the test sample, will bind to the
SPN, forming a protein:SPN complex. This protein:SPN complex can be
detected by a detection sensor during a process of the test.
[0253] In some embodiments, detection agents for eight major food
allergens (i.e. wheat, egg, milk, peanuts, tree nuts, fish,
shellfish and soy) may be provided as disposables. In one aspect,
constructs of the detection agents may be stored with MgCl.sub.2,
or buffer doped with KCl. MgCl.sub.2 keeps constructs closed
tightly, while KCl opens them slightly for bonding.
[0254] In some embodiments, the detection sensor is a nucleic acid
printed solid substrate. As used herein, the term "detection
sensor" refers to an instrument that can capture a reaction signal,
i.e. the reaction signal derived from the binding of allergen
proteins and detection agents, measure a quantity and/or a quality
of a target, and convert the measurement to a signal that can be
measured digitally.
[0255] In some embodiments, the detection sensor is a solid
substrate, such as a glass chip, coated with nucleic acid molecules
(as referred to herein as nucleic acid chip or DNA chip). For
example, the detection sensor may be the glass chip 333 inserted
into the reaction chamber 331 of the present disclosure or a
chipannel 710 in the test cup 300 (FIG. 7A). The detection sensor
may also be a separate glass chip, for example, prepared from glass
wafer and soda glass, or a microwell, or an acrylic glass, or a
microchip, or a plastic chip made of COC (cyclic olefin copolymer)
and COP (cyclo-olefin polymer), or a membrane like substrate (e.g.,
nitrocellulose), of which the surface is coated with nucleic acid
molecules.
[0256] In some embodiments, the nucleic acid coated chip may
comprise at least one reaction panel and at least two control
panels. The reaction panel is printed with nucleic acid probes that
hybridize to the SPN. As used herein, the term "nucleic acid probe"
refers to a short oligonucleotide comprising a nucleic acid
sequence complementary to the nucleic acid sequence of a SPN. The
short complementary sequence of the probe can hybridize to the free
SPN. When the SPN is not bound by a target allergen, the SPN can be
anchored to the probe through hybridization. When the SPN bind to a
target allergen to form a protein:SPN complex, the protein:SPN
complex prevents the hybridization between the SPN and its nucleic
acid probe.
[0257] In some examples, the probe comprises a short nucleic acid
sequence that is complementary to the sequence of the 3' end of the
SPN that specifically binds to a target allergen protein. In this
context, the SPN specific to the target allergen protein is
provided in the extraction/homogenization buffer. When the sample
is processed in the homogenization chamber 321, the target
allergen, if present in the test sample, will bind to the SPN, and
form a protein:SPN complex. When the sample solution flows to the
detection sensor, e.g., the DNA chip 333 in the reaction chamber
331 (FIG. 3B) or the chipannel 710 (FIG. 7A), the bound allergen
protein prevents the SPN from hybridizing to the complementary SPN
probes on the chip surface. The protein:SPN complex is washed off
and no fluorescence signal is detected. In the absence of the
target allergen proteins in the test sample, the free SPN will bind
to the complementary SPN probes on the chip surface. A fluorescence
signal will be detected from the reaction panel (as shown in FIGS.
13A and 13B).
[0258] In some embodiments, the detection sensor, e.g., nucleic
acid printed chip, further comprises at least two control panels.
The control panels are printed with nucleic acid molecules that do
not bind to a SPN or a protein (referred herein as "control nucleic
acid molecules"). In some examples, the control nucleic acid
molecules are labeled with a fluorescence marker.
[0259] In some embodiments, nucleic acid probes may be printed to a
reaction panel at the center of a glass chip ("unknown") and
control nucleic acid molecules may be printed to the two control
panels at each side of the reaction panel on the glass chip, as
illustrated in FIG. 13A.
[0260] In some embodiments, the nucleic acid chip (DNA chip) may be
prepared by any known DNA printing technologies known in the art.
In some embodiments, the DNA chip may be prepared by using single
spot pipetting to pipette nucleic acid solution onto the glass
chip, or by stamping with a wet PDMS stamp comprising a nucleic
acid probe solution followed by pressing the stamp against the
glass slide, or by flow with microfluidic incubation chambers.
[0261] As a non-limiting example, a glass wafer can be laser cut to
produce 10.times.10 mm glass "chips". Each chip contains three
panels: one reaction panel (i.e. the "unknown" area in the chip
demonstrated in FIG. 13A) that is flanked by two control panels
(FIG. 13A). The reaction panel contains covalently bound short
complementary nucleic acid probes to which SPNs specific to an
allergen protein bind. The SPNs are derived from aptamers and
modified to contain a CY5 fluorophore. In the absence of the target
allergen protein, SPNs are free to bind to the probes in the
reaction panel, resulting in a high fluorescence signal. In the
presence of the target allergen protein, the SPN:probe hybridizing
interface is occluded by the binding of the target protein to the
SPNs, thereby resulting in a decrease in fluorescence signal on the
reaction panel. In a detection assay, the reaction panel of the
chip faces a small reaction chamber (e.g. the reaction chamber 331)
flanked by an inlet and outlet channel (e.g., 336 in FIG. 3H) of
the cartridge (e.g., the cup 300). During food homogenization, the
SPN in the extraction buffer binds to the target allergen if it is
present in the sample forming a protein:SPN complex. The processed
sample solution including the protein:SPN complex enters the
reaction chamber 331 via the inlet, through fluidic movement driven
by a vacuum pump. The solution then exits into a waste chamber 323
via the outlet channel. After exposure to the sample, the reaction
panel is then washed, revealing a fluorescence signal with an
intensity correlated to the target allergen concentration.
[0262] In some embodiments, the wash buffer is optimized to improve
wash efficiency, increasing baseline signal and decreasing
non-specific binding. As a non-limiting example, the wash buffer
may be an optimized PPB buffer, including pluronic F-127 (e.g., 2%
w/v), PEG-8000 (2% w/v), Brij 58 (e.g., 0.2% w/v) and EPPS (e.g.,
20 mM), pH8.4.
[0263] In accordance with the present disclosure, the two control
panels are constantly bright areas on the chip sensor that produce
a constant signal as background signals 1301 and 1302 (FIG. 13B).
In addition, the two control panels compensate for laser
illumination and/or disposable cartridge misalignment. If the
cartridge is perfectly aligned, then the fluorescence background
signals 1301 and 1302 would be equal (as shown in FIG. 13B). If the
measured control signals are not equal, then a look-up table of
correction factors will be used to correct the unknown signal as a
function of cartridge/laser misalignment. The final measurement is
a comparison of the signal 1303 of the unknown test area against
the signal levels of the control areas. The comparison level may be
one of the lot-specific parameters for the test.
[0264] Food samples with high background fluorescence measurements
from the reaction area may produce a false negative result. A
verification method may be provided to adjust the process.
[0265] The final fluorescence measurement of the reaction panel,
after being compared to the controls and any lot specific
parameters may be analyzed and a report of the result may be
provided.
[0266] Accordingly, the light absorption and light scattering
signals may also be measured at the baseline level, before and/or
after the injection of the processed food sample. These
measurements will provide additional parameters to adjust the
detection assay. For example, such signals may be used to look for
residual food in the reaction chamber 331 after wash.
[0267] In addition to the parameters discussed above, one or more
other lot-specific parameters may also be measured. The
optimization of the parameters, for example, may minimize the
disparity in the control and unknown signal levels for the
chips.
[0268] In some embodiments, the monitoring process may be automatic
and is controlled by a software application. Evaluation of the DNA
chip and test sample, the washing process and the final signal
measurement may be monitored during the detection assay.
[0269] Allergen families that can be detected using the detection
system and device described herein include allergens from foods,
the environment or from non-human proteins such as domestic pet
dander. Food allergens include, but are not limited to proteins in
legumes such as peanuts, peas, lentils and beans, as well as the
legume-related plant lupin, tree nuts such as almond, cashew,
walnut, Brazil nut, filbert/hazelnut, pecan, pistachio, beechnut,
butternut, chestnut, chinquapin nut, coconut, ginkgo nut, lychee
nut, macadamia nut, nangai nut and pine nut, egg, fish, shellfish
such as crab, crawfish, lobster, shrimp and prawns, mollusks such
as clams, oysters, mussels and scallops, milk, soy, wheat, gluten,
corn, meat such as beef, pork, mutton and chicken, gelatin,
sulphite, seeds such as sesame, sunflower and poppy seeds, and
spices such as coriander, garlic and mustard, fruits, vegetables
such as celery, and rice. The allergen may be present in a flour or
meal, or in any format of products. For example, the seeds from
plants, such as lupin, sunflower or poppy can be used in foods such
as seeded bread or can be ground to make flour to be used in making
bread or pastries.
Applications
[0270] The detection systems, devices and methods described herein
contemplate the use of nucleic acid-based detector molecules such
as aptamers for detection of allergens in food samples. The
portable devices allow a user to test the presence or absence of
one or more allergens in food samples. Allergen families that can
be detected using the device described herein include allergens
from legumes such as peanuts, tree nuts, eggs, milk, soy, spices,
seeds, fish, shellfish, wheat gluten, rice, fruits and vegetables.
The allergen may be present in a flour or meal. The device is
capable of confirming the presence or absence of these allergens as
well as quantifying the amounts of these allergens.
[0271] In a broad concept, the detection systems, devices and
methods described herein may be used for detection of any protein
content in a sample in a large variety of applications in addition
to food safety, such as, for example, medical diagnosis of diseases
in civilian and battlefield settings, environmental
monitoring/control and military use for the detection of biological
weapons. In even broad applications, the detection systems, devices
and methods of the present disclosure may be used to detect any
biomolecules to which nucleic acid-based detector molecules bind.
As some non-limiting examples, the detection systems, devices and
methods may be used on the spot detection of cancer markers,
in-field diagnostics (exposure the chemical agents, traumatic head
injuries etc.), third-world applications (TB, HIV tests etc.),
emergency care (stroke markers, head injury etc.) and many
others.
[0272] As another non-limiting example, the detection systems,
devices and methods of the present disclosure can detect and
identify pathogenic microorganisms in a sample. Pathogens that can
be detected include bacteria, yeasts, fungi, viruses and virus-like
organisms. Pathogens cause diseases in animals and plants;
contaminate food, water, soil or other sources; or is used as
biological agents in military fields. The device is capable of
detecting and identifying pathogens.
[0273] Another important application includes the use of the
detection systems, devices and methods of the present disclosure
for medical care, for example, to diagnose a disease, to stage a
disease progression and to monitor a response to a certain
treatment. As a non-limiting example, the detection device of the
present disclosure may be used to test the presence or absence, or
the amount of a biomarker associated with a disease (e.g. cancer)
to predict a disease or disease progression. The detection systems,
devices and methods of the present disclosure are constructed to
analyze a small amount of test sample and can be implemented by a
user without extensive laboratory training.
[0274] Other expanded applications outside of the field of food
safety include in-field use by military organizations, testing of
antibiotics and biological drugs, environmental testing of products
such as pesticides and fertilizers, testing of dietary supplements
and various food components and additives prepared in bulk such as
caffeine and nicotine, as well as testing of clinical samples such
as saliva, skin and blood to determine if an individual has been
exposed to significant levels of an individual allergen.
EQUIVALENTS AND SCOPE
[0275] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments in accordance with the
disclosure described herein. The scope of the present disclosure is
not intended to be limited to the above Description, but rather is
as set forth in the appended claims.
[0276] A number of possible alternative features are introduced
during the course of this description. It is to be understood that,
according to the knowledge and judgment of persons skilled in the
art, such alternative features may be substituted in various
combinations to arrive at different embodiments of the present
disclosure.
[0277] Any patent, publication, internet site, or other disclosure
material, in whole or in part, that is said to be incorporated by
reference herein is incorporated herein only to the extent that the
incorporated material does not conflict with existing definitions,
statements, or other disclosure material set forth in this
disclosure. As such, and to the extent necessary, the disclosure as
explicitly set forth herein supersedes any conflicting material
incorporated herein by reference. Any material, or portion thereof,
that is said to be incorporated by reference herein, but which
conflicts with existing definitions, statements, or other
disclosure material set forth herein will only be incorporated to
the extent that no conflict arises between that incorporated
material and the existing disclosure material.
[0278] In the claims, articles such as "a," "an," and "the" may
mean one or more than one unless indicated to the contrary or
otherwise evident from the context. Claims or descriptions that
include "or" between one or more members of a group are considered
satisfied if one, more than one, or all of the group members are
present in, employed in, or otherwise relevant to a given product
or process unless indicated to the contrary or otherwise evident
from the context. The disclosure includes embodiments in which
exactly one member of the group is present in, employed in, or
otherwise relevant to a given product or process. The disclosure
includes embodiments in which more than one, or the entire group
members are present in, employed in, or otherwise relevant to a
given product or process.
[0279] It is also noted that the term "comprising" is intended to
be open and permits but does not require the inclusion of
additional elements or steps. When the term "comprising" is used
herein, the term "consisting of" is thus also encompassed and
disclosed.
[0280] Where ranges are given, endpoints are included. Furthermore,
it is to be understood that unless otherwise indicated or otherwise
evident from the context and understanding of one of ordinary skill
in the art, values that are expressed as ranges can assume any
specific value or subrange within the stated ranges in different
embodiments of the disclosure, to the tenth of the unit of the
lower limit of the range, unless the context clearly dictates
otherwise.
[0281] In addition, it is to be understood that any particular
embodiment of the present disclosure that falls within the prior
art may be explicitly excluded from any one or more of the claims.
Since such embodiments are deemed to be known to one of ordinary
skill in the art, they may be excluded even if the exclusion is not
set forth explicitly herein. Any particular embodiment of the
compositions of the disclosure (e.g., any antibiotic, therapeutic
or active ingredient; any method of production; any method of use;
etc.) can be excluded from any one or more claims, for any reason,
whether or not related to the existence of prior art.
[0282] It is to be understood that the words which have been used
are words of description rather than limitation, and that changes
may be made within the purview of the appended claims without
departing from the true scope and spirit of the disclosure in its
broader aspects.
[0283] While the present disclosure has been described at some
length and with some particularity with respect to the several
described embodiments, it is not intended that it should be limited
to any such particulars or embodiments or any particular
embodiment, but it is to be construed with references to the
appended claims so as to provide the broadest possible
interpretation of such claims in view of the prior art and,
therefore, to effectively encompass the intended scope of the
disclosure.
EXAMPLES
Example 1: Testing Filter Materials and Filtering Efficiency
[0284] Various filter materials and their combinations are tested
for filtering efficiency and effect on signal measurement, for
example, the loss of detection agents (SPNs). Commercially
available filter materials such as membranes (PES, glass fiber,
PET, PVDF, etc.), cotton, sand, mesh and silica are tested.
[0285] A filter including a combination of different filter
materials is assembled. In one example, the filter assembly is
composed of cotton and glass filter with a pore size of 1 .mu.m.
The cotton depth filter and paper filter are constructed to filter
the sample sequentially. The filter assembly is tested for
filtering different food matrices. The recovery of proteins and
SPNs during the filtering process is measured. Various cotton
volumes are used to construct the depth filters and the cotton
depth filters are combined with membrane filters. These filter
assemblies are tested for filtration efficiency and SPN recovery.
In one study, 0.5 g of a food sample is collected and homogenized
in 5 ml EPPS buffer (pH 8.4) (Tween 0.1%) and the homogenized food
sample is incubated with 5 nM SPNs (signaling polynucleotides)
labeled with Cy5 that is specific to an allergen protein. After
incubation, a portion of the mixture is run through the filter
assemblies and the recovery of proteins and SPNs is measured and
compared with the pre-filtering measurements.
[0286] The filters are further tested and optimized to ensure
efficiency of filtration and avoidance of significant SPN loss. In
addition to testing different filter materials and their
combinations, other parameters such as pore sizes, filtering areas
(e.g., surface area/diameter, height of the depth filter),
filtering volumes, filtration time and pressure required to drive
the filtering process, etc., are also tested and optimized for
various food matrices.
[0287] In one study, bleached cotton balls are used to assemble the
depth filters with different filter volumes. Cotton filters with
different ratios of width (i.e. diameter) and height are
constructed; each model has a ratio of width and height ranging
from about 1:30 to about 1:5. The cotton depth filters are then
tested for filtration efficiency with different food masses and
buffer volumes. In another study, these model cotton filters are
assembled together with a PET membrane filter with 1 .mu.m pore
size and about 20 mm.sup.2 filtrating area. Various food samples
are homogenized and filtered through each filter assembly using
different volumes of buffer. The filtrates are collected and the
percentage of recovery is compared for each condition.
[0288] In another study, food samples are spiked with or without 50
ppm peanut. The spiked samples are homogenized, for example using
the rotor 340 (e.g., as illustrated in FIGS. 3B and 3C) and the
extractions are mixed with SPNs that specifically bind to peanut
allergen. The SPN contains a Cy5 label at the 5' end of the
sequence. The mixture is filtered through a depth filter (e.g., a
depth filter made of cotton) and a membrane filter (pore size: 1
.mu.m). Fluorescence signals are measured and compared with the
measurements of the pre-filtered mixture.
[0289] In separate studies, several parameters of each filter
assembly are tested and measured including the pressure and time
required for filtering, protein and nucleic acid binding, washing
efficiency and assay compatibility and sensitivity. The assay
compatibility is measured as the baseline intensity.
Example 2: MgCl.sub.2 Formulations
[0290] Several solid MgCl.sub.2 formulations were tested to replace
the addition of MgCl.sub.2 solution after the sample homogenization
in extraction buffer. The following characteristics of each
formulation tested are evaluated: (1) the time to dissolve; (2) the
final concentration of dissolved MgCl.sub.2; (3) the effect of
additives in the formulations on the detection assay; (4) no
agitation required to dissolve; and (5) no breakup into powder and
not blocking the outlet of the homogenization chamber.
Lyophilized MgCl.sub.2 Formulation
[0291] 34 MgCl.sub.2 formulations were lyophilized in 1.5 mL
Eppendorf tubes and tested for dissolution time, mechanical
stability, exposure to the extraction buffer for 10 seconds without
agitation, and other features. 2 formulations are rapidly
dissolving and do not form powder. Several MgCl.sub.2 formulations
were exposed to the extraction buffer for 10 seconds without
agitation and the magnesium content in the recovered buffer was
determined by a BioVision Magnesium assay and the assay as
described herein. The assay results indicate that the lyophilized
MgCl.sub.2 formulation comprising maltodextrin and
hydroxyethylcellulose (HEC) (Table 1) gives the highest intensity
of SPNs in buffer as shown in FIG. 16A.
MgCl.sub.2 as a Filter Component
[0292] MgCl.sub.2 formulations (Table 1) were deposited on a cotton
filter and dried at 60.degree. C. The extraction buffer was pulled
through the cotton filter with 1 psi vacuum. The percentage of
magnesium recovered in filtrate was measured by the BioVision
colorimetric magnesium assay. The MgCl.sub.2 formulation comprising
maltodextrin and hydroxyethylcellulose (HEC) (Table 1) was compared
with what was recovered in MgCl.sub.2 solution and MgCl.sub.2 on
the filter (FIG. 16B).
MgCl.sub.2 as Film
[0293] 10 different MgCl.sub.2 formulations were deposited on
polystyrene supports and cured. The dissolution time was measured
and all formulations dissolved in 10 seconds. The results indicate
that none of the formulations have a strong adhesion to the
polystyrene support.
TABLE-US-00001 TABLE 1 Components of MgCl.sub.2 formulations
Formulations containing 1.0% glycerol glycerol 1.0% PEG 2.00% PEG
1.00% PEG 0.3% PEG 0.5% glycine 2.5% sugar 0.5% maltodextrin 0.5%
PEG 0.3% Formulations containing 0.7% glycerol glycerol 0.7% PEG
2.00% PEG 1.00% PEG 0.3% PEG 0.5% glycine 2.5% sugar 0.5%
maltodextrin 0.5% PEG 0.3% Formulations containing 0.5% glycerol
glycerol 0.5% PEG 2.00% PEG 1.00% PEG 0.3% PEG 0.5% glycine 2.5%
sugar 0.5% maltodextrin 0.5% PEG 0.3% PEG 2.0% glycine 2.5% PEG
5.0% glycine 2.5% maltodextrin 0.5% HEC 0.1%
[0294] Based on the test results, several fast-dissolving solid
MgCl.sub.2 formulations are selected (as shown in Table 2). The
dissolution time for the filter deposition is dependent on flow
rate. When the fastest flow rate was tested, the solid formulation
dissolved in 10 seconds (as shown in Table 2).
TABLE-US-00002 TABLE 2 Fast-dissolving and mechanically robust
solid MgCl.sub.2 formulations Lyophilized Incurred in pellet Film
filter Leading 0.5% glycerol/ 1% maltodextrin/ 1% maltodextrin/
formulation 0.5% sucrose 0.1% hydroxyethyl 0.1% hydroxyethyl
cellulose cellulose Time for 12 Seconds 16 seconds 10 seconds
resuspension Stability - + N/A following agitation (vortex 1
minute) Mg recovery 100% 100% 80% in 10 seconds (compared to
MgCl.sub.2 solution)
* * * * *